Trends in Analytical Chemistry 167 (2023) 117274

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Trends in Analytical Chemistry

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Analyses of chemical components and their functions in single species

plant-derived exosome like vesicle
Sisi Zhou a, Yu Cao a, Fanshu Shan a, Puzhen Huang a, Yao Yang b, **, Songqin Liu a, *
a
Jiangsu Provincial Key Laboratory of Critical Care Medicine, Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Key Laboratory of
Environmental Medicine Engineering, Ministry of Education, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China
b
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210023, China

A R T I C L E I N F O A B S T R A C T

Keywords: Plant-derived exosome like vesicles (PDEVs) are small vesicles released by a variety of plants including fruits and
Chemical component vegetables. Like the plants they derived from, PDEVs have shown multiple health benefits, such as modulating
Health functionality gut microbiome and homeostasis, promoting intestinal tissue regeneration, and exerting anti-tumor and anti-
Omics analysis
inflammatory effects. Depending on its plant species, each PDEV has different components (lipids, proteins,
Plant-derived exosome like vesicle
Single species
nucleic acid, and other small active metabolites), thus its intrinsic molecules exhibit distinct signaling pathway
regulation modes through various mechanisms. In this review, we summarize recent advances in identification of
the chemical components in single species PDEV, especially by omics analysis. Herein, single species PDEV refers
to PDEVs from the same plant. Beyond that, the focus is laid on the health-beneficial applications of different
species PDEVs. The challenges and their potency as new tools for next-generation biotherapeutic approaches are
also discussed.

1. Introduction plants [11]. To date, Plant EVs have been found in many plant species,
such as lemon [12], broccoli [13], ginger, grapefruit, blueberry, etc.
Exosomes are extracellular vesicles (EVs) released by various cells [14]. In fact, current plant EV literatures contain confusing diversity of
with a size of 30–150 nm. They were originally thought to be one type of terms, such as ‘plant-derived exosome like nanoparticles’ [15,16],
carriers through which cells are able to dispose their residues and un­ ‘plant-derived vesicle-like nanoparticles’ [17], ‘plant-derived exosomes’
necessary materials. At present, they are considered as functional vesi­ [18], ‘plant-derived edible nanoparticles’ [19]. Although the
cles because they carry many bioactive compounds such as proteins [1], morphology (nanosized vesicles), compositions (lipids, RNAs, and pro­
lipids [2], and nucleic acids [3,4]. They participate in the intercellular teins) and even biofunctions (participating in the intercellular commu­
communications of various physiological and pathological processes nication between species) of these plant EVs are similar to mammalian
such as immune response [5], signal transduction, antigen presentation exosomes, it should be emphasized that the biogenesis and secretion
[6], etc. Almost all eukaryotic cell-released EVs, chemical components pathways of plant EVs investigated so far are still unclear and are
of the exosomes are greatly dependent on the type or functional state of therefore not suitable to be called exosomes [20]. Hence, they are
the parent cells. In recent years, upon extensive research, mammalian named plant-derived exosome like vesicles (PDEVs) in this review.
exosomes have become a hotspot in various scientific research fields Current studies show that PDEVs can be used for intercellular
including clinical diagnostics, therapeutics, and drug deliveries [7–9]. communication and immune regulation to resist the invasion of patho­
With a growing appreciation of the importance of EVs in mammals, gens in plants [21]. Meanwhile, PDEVs are one type of potential func­
the interest in plant EVs has increased over the last few years. In fact, the tional components with multiple health benefits. For example, they can
first study of plant EVs was in 1967, which was 15 years earlier than that regulate the gene/protein expression of recipient cells, modulate gut
of mammalian exosomes [10]. Several lines of evidence support that the microbiome and homeostasis, promote intestinal tissue regeneration,
fusion of multivesicular bodies (MVBs) with the plasma membrane and exert anti-tumor and anti-inflammatory effects with their specific
might result in the release of vesicles into the extracellular space in chemical components (e.g., lipids, proteins, nucleic acids, or

* Corresponding author.
** Corresponding author.
E-mail addresses: [email protected] (Y. Yang), [email protected] (S. Liu).

https://doi.org/10.1016/j.trac.2023.117274
Received 29 May 2023; Received in revised form 24 August 2023; Accepted 3 September 2023
Available online 7 September 2023
0165-9936/© 2023 Elsevier B.V. All rights reserved.
S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

Fig. 1. Schematic representation of PDEVs chemical components. Abbreviation: PA, phosphatidic acid; HSPs, heat shock proteins.

metabolites) (Fig. 1) [22]. Depending on its plant origin, PDEVs have Nevertheless, the bioactivities between PDEVs and the plant extracts
different components, thus its intrinsic molecules exhibit distinct from same species are different [30]. It is also noteworthy that the
signaling pathway regulation modes through various mechanisms [23]. chemical composition profiles of PDEVs maintain significant discrep­
Yun Teng et al. showed that the lipid composition of PDEVs from ginger ancies from those of mammalian exosomes, especially metabolites,
determines their uptake by specific gut bacteria [24]. Gabriella Pocsfalvi which are abundant in PDEVs but rare in mammalian exosomes [31,32].
et al. identified 600–800 proteins from citrus fruit-derived vesicles, Thus, to promote the biomedical applications of PDEVs, it is important
which contains various membrane transporters, intra-vesicular trans­ to isolate and identify the active ingredients of different species PDEVs,
porters, and a large number of enzymes (e.g., hydrolases and oxidore­ and verify the functions of the ingredients. With the rapid development
ductases) [25]. The microRNA (miRNA) mir-5781 in PDEVs derived of high-throughput technologies, ‘Omics’-based approaches such as
from soybean can directly target interleukin-17a (IL-17A) and plays an lipidomics, proteomics, transcriptomics, and metabolomics, have gained
important role in inflammatory response [26]. Besides the inherent a lot of popularity among researchers. They have been developed as
biological activities, PDEVs can act as excellent nanocarriers for deliv­ effective methods for whole characterization of chemical components in
ering various therapeutic compounds [27]. PDEVs extracted from or­ single species PDEV.
ange (Citrus sinensis) juice were investigated as carriers for SARS-CoV-2 It’s worth noting that the precondition for the identification and
mRNA vaccine [28]. It was also reported that leukocyte functional study of different parts of single species PDEV is to obtain
function-associated antigen-1 (LFA-1) and CXC chemokine receptor 2 PDEVs with high purity and quality. Several separation methods have
(CXCR2) coated on the surface of grapefruit PDEVs could realize tumor been used to concentrate PDEV of single species, such as ultracentrifu­
target specificity [29]. It is noteworthy that the therapeutic role is not gation combined with density gradient centrifugation (Fig. 2) [33–36],
only dependent on the delivered drugs but also the selection of PDEVs. co-precipitation [37–39], immunoaffinity capture-based techniques
Therefore, identification of active constituents in PDEVs is crucial to [40], and microfluidic-based separation methods [41–43]. Among them,
understand the specific impact of different species PDEVs on health and ultracentrifugation combined with density gradient centrifugation is the
disease development, as well as the selection of a certain species PDEV main and widely used method. Specifically speaking, the plants need to
as delivery system. In this review, we attempt to summarize the iden­ be washed and juiced with simple processing first. Then, the juice is
tification of chemical components in single species PDEV, and discuss centrifuged at 2000 g and 10,000 g to remove dead cells and cell debris,
the potential applications of these chemical components for human respectively. Sometimes the supernatant is subjected to pass through a
health. sterilizing grade filter membrane (0.2 or 0.45 μm) before ultracentri­
fugation to remove large vesicles [33,34]. Subsequently, the supernatant
2. Identification of chemical components in single species PDEV is ultracentrifuged at 100,000 g for 90 min to precipitate crude PDEVs.
Centrifugation conditions (speed and time) should be optimized ac­
PDEVs are naturally generated and carry versatile components cording to the specific nature of plant species. Crude PDEVs isolated by
(proteins, lipids, genetic materials, and metabolites) from plant cells, ultracentrifugation alone contain both PDEVs and biological aggregates
some of which have been recently proven to be able to treat specific (protein or nucleic acid contaminants). Thus, as shown in Fig. 2, dif­
diseases or maintain healthy body functions (Table 1). This might ferential centrifugation is often combined with density gradient centri­
attribute to that the multiple components in original plants are selec­ fugation to further purify PDEVs. The most widely used gradient
tively encapsulated in PDEVs and the PDEVs have high bioavailability. medium is sucrose [33–36], and the commonly used gradient solutions

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S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

Table 1 Table 1 (continued )

List of the main components of single species PDEV and their biological Active Plant species Functions Ref.
functions. components
Active Plant species Functions Ref. (AMPK)-mediated induction of dendritic
components cells tolerance
Lipids Orange Reduce plasma lipids and inflammation [60] Ginger 6-gingerol and 6-shogaol [51]
in gastrointestinal diseases Participate in the reduction of acute
Ginger Phosphatidic acid (PA) (34:2) [71] colitis, enhancement of intestinal repair,
Selectively bind to hemin-binding and prevention of colitis-associated
protein 35 (HBP35) on the surface of cancer
P. gingivalis and inhibit its pathogenicity Turmeric Curcumin [133]
Grapefruit Phosphatidylcholine (PC) [24] Regulate the expression of the pro-
Preferentially be taken up by inflammatory cytokines and antioxidant
Ruminococcaceae sp. (TSD-27) gene or mediate inactivation of the NF-
Ginseng Diacylglycerol [45] kappaB pathway
Exhibit anti-senescence and anti- Orange Carbohydrate (glucose, fructose, [60]
pigmentation effects by activating sucrose) and amino acid
protein kinase C (PKC) signaling and Reverse diet-induced gut modifications
increasing the expression of HMGB1 and treat obesity-associated intestinal
Ceramide [73] complications
Alter macrophage polarization via Toll- Grapefruit Organic acid (glycolic and citric acids) [61]
like receptor-4 activation to inhibit Exhibit anticancer properties
melanoma growth Cannabis Cannabidiol [133]
Arabidopsis Glycosylinositolphosphoceramide [76] Induce cell death by activating
Promote the secretion of EVs and ROS mitochondrial-dependent apoptosis
burst signaling pathways in hepatocellular
Celery Fatty acids [65] carcinoma cell lines
Suppress and regulate the immune Tea leaf Polyphenol and flavonoid [128]
response Induce apoptosis of tumor cells
Proteins Citrus Clathrin [81, Pueraria Puerarin [131]
Is related to vesicle formation and 84] lobata Shift M1 macrophages toward the M2
transport and enhance anti-inflammatory effects
Patellin-3-like protein Citrus limon, Vitamin C and glutathione [126,
Promote the transfer of PDEVs between strawberry Protect cells against oxidative stress 127]
different cellular compartments
Garlic II lectin [83]
Bind to CD98 on the HepG2 cells and are 8%, 30%, 45%, and 60% sucrose. Mostly, sucrose gradients of the
exhibit in vitro anti-inflammatory effect 30%/45% layer are harvested as the preeminent source of PDEVs, but
Mulberry Heat shock protein family A member 8 [86] PDEVs from ginseng are isolated at concentrations between 8 and 30%
(HSPA8)
Target intestinal epithelial cells, and
concentration [33]. Another gradient medium is iodixanol with gradi­
prevent colitis in a mouse model ents of 10%, 20% and 30%, respectively. Most of the PDEVs accumulate
Citrus Catalase (CAT), superoxide dismutase [24, at iodixanol gradient of 20% and 83% with diameters of 30–100 nm
(SOD) 84] [34]. As a gold standard method for the isolation of PDEVs, ultracen­
Protect cells from oxidative damage by
trifugation has several advantages, such as wide adjustability, low cost
scavenging reactive oxygen species
(ROS). and contamination risks, and large sample capacity. After further den­
RNAs Ginseng Promote neural recovery by up- [56] sity gradient centrifugation, the purity of PDEVs is improved while their
regulating the phosphatidylinositol 3 yields decreases significantly. Other separation methods and their fea­
kinase (PI3K) signaling pathway tures have been summarized in many reviews and will not be discussed
Coffee, Target human apoptosis-related target [109]
moringa genes, promote apoptosis of the tumor
here [44]. In general, the methods and conditions for the separation of
oleifera cells single species PDEV should be selected according to their plant sources.
Bitter melon Mediate the anti-inflammatory activity [26] Nevertheless, it is reported that the plants need to be grinded in a
of target cells by regulating the mixer/blender, releasing not only EVs, but also native intracellular
expression of NLRP3 mRNA
vesicles when their cells are broken open. Thus, unlike mammalian
Ginger Mdo-mir7267-3p [24]
Target ycnE gene in Lactobacillus exosomes, PDEVs obtained with the destructive processes contain true
rhamnosus and induce production of plant EVs and intracellular vesicles.
indole-3-carboxaldehyde, ameliorate
mouse colitis
Ginger, Aly-miR396a-5p- and rlcv-miRrL1-28- [112] 2.1. Identification of lipids in single species PDEV
Momordica 3p-
Charania Target Nsp12 gene of the lung epithelial
Similar to mammalian exosomes, each PDEV has a lipid bilayer. The
cells exposed to severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) structure and content of the lipid bilayer are essential in the interaction
and inhibit lung inflammation between PDEVs and mammalian cells. Lipidomics is a mass spectrom­
Novel 18, novel 20, and novel 27 [51, etry (MS)-based technology to map the whole lipidomic profiles or
Are involved in immunomodulatory or 113] identify specific lipids in PDEVs from different plant species. Prior to MS
metabolic regulation
miRNA5266 [118]
lipidomic analysis, total lipids of single species PDEV are extracted using
Attenuate ischemia-reperfusion-induced the Bligh and Dyer method or the Folch method [45,46]. Briefly, PDEVs
damage to the blood brain barrier and are mixed with chloroform: methanol (2 : 1, v/v) and vortexed. Then the
inhibit neuronal apoptosis same amount of chloroform and double desterilized water are added
Metabolites Broccoli Sulforaphane [13]
sequentially. Then the mixture is shaked well, centrifuged to obtain two
Prevent mouse colitis by adenosine
monophosphate-activated protein kinase phases (aqueous phase and organic phase). The organic phase is aspi­
rated and dried, followed by resuspending in chloroform for lipid
composition analysis with triple quadrupole MS. The data are reported

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S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

Fig. 2. Identification of chemical components in different species PDEVs. Various fractions are isolated with density gradient centrifugation method. Abbreviation:
UC, ultracentrifugation.

as percentage of total signal of the molecular species after normalization proteins, and removal of low molecular weight impurities (e.g., de­
of the signals to internal standards of the same lipid class. To explore the tergents and buffer components) which are detrimental for MS analysis.
biofunction of each lipid in PDEVs, thin-layer chromatography (TLC) Thus, it is useful to combine the in-gel method with in-solution prote­
analysis is used to separate PDEVs’ lipids in chloroform. Each band omics analysis to improve protein identification efficiency [54]. Today,
containing different lipid is identified according to the standard samples MS-based shotgun proteomics is becoming a powerful and broadly
in TLC plate and then collected for high-performance liquid chroma­ applicable technology to study protein on a large scale. From which, a
tography (HPLC) analysis [24]. global picture of biofunctions of proteins in single species PDEV is ob­
tained. However, it is still challenging for this technology to realize the
2.2. Identification of proteins in single species PDEV identification of all proteins in PDEVs because different species plants
require different sets of protein databases for matching.
Currently, some transmembrane proteins, such as CD63, CD81 and
CD9, have been identified as possible markers of mammalian cell- 2.3. Identification of nucleic acids in single species PDEV
derived exosomes. They are involved in cellular communication in a
variety of physiological and pathological processes [47–49]. Although Besides proteins, many nucleic acids such as DNA, siRNA, and
the concentrations of the proteins detected in PDEVs are low, they still miRNA are also identified in PDEVs. Polymerase chain reaction (PCR)
play important roles in the biological functions of PDEVs. However, the and sequencing-based technology can efficiently identify the types and
functional protein markers of single species PDEV are not well under­ components of nucleic acids in PDEVs [55]. Sequencing-based technique
stood and require further investigation. The commonly used protein involves several major steps such as the preparation of total RNAs, the
analysis methods are colorimetric assays, such as bicinchoninic acid construction and sequencing of small RNA library, and the prediction
(BCA), fluorimetric assays (e.g., enzyme linked immunosorbent assay, and functional annotation of miRNA target genes [24,26,56]. In brief,
ELISA), sodium dodecyl sulfate (SDS)-polyacrylamide gel electropho­ total RNAs of single species PDEV are obtained with Trizol reagent.
resis (PAGE), and western blotting [50]. However, complete profiling of Then, the quality and quantity of resultant RNAs are examined by 1%
the proteins in single species PDEV is quite challenging for these agarose gel electrophoresis and a NanoDrop Spectrophotometer. To
methods. To address this, shotgun proteomics analysis was developed construct library, the RNAs are fragmented into small pieces ranging
using high-resolution, high-mass accuracy MS equipped with a nano­ from 14 to 36 nt in size, reverse-transcribed to create the cDNA, and
spray source, such as Bruker Maxis II mass spectrometer (Bruker Dal­ amplified with PCR. At last, the paired-end sequencing is performed on
tonics GmbH) and LTQ Orbitrap mass spectrometer (Thermo Fisher) an Illumina Hiseq 4000 platform. The resulting sequence reads are
[25,51]. This method requires a complex sample preparation process filtered to remove low-quality reads, repeat sequences, and adaptor se­
including protein extraction, protein reduction and alkylation, trypsin quences. The mapped reads are used to look for known miRNAs using
digestion, and peptide desalting [52]. Briefly, proteins from single spe­ the miRBase database (http://www.mirbase.org/index.shtml). Tar­
cies PDEV are isolated by suspending PDEVs in lysis buffer, followed by getScan is used to annotate miRNA target gene [57]. Functional analysis
sonication and centrifugation to eliminate PDEV debris. Then the su­ of target genes is achieved using the Gene Ontology (GO) and Kyoto
pernatant containing proteins is collected and protein concentration is Encyclopedia of Genes and Genomes (KEGG) pathway analyses [58].
measured with Bradford or BCA protein assay. Before digestion, the The next generation sequencing-based transcriptomics is a powerful
quality of the protein samples is controlled with SDS-PAGE analysis. approach to thoroughly characterize the nucleic acids of single species
Then, proteins are reduced with DL-dithiothreitol, alkylated with PDEV, which could reveal the molecular mechanism of PDEV-mediated
iodoacetamide, and digested with MS grade trypsin. Several strategies physiological and physiopathological processes. However, since
have been used to identify more proteins. For example, by creating pre-miRNAs sequences are not available for certain plants, the identi­
complementary peptides, combinational use of highly selective pro­ fication of miRNAs from those plants is still a difficult task to complete
teases was employed to improve the identification coverage and sensi­ [23].
tivity of proteins and proteomes. Fractionation of peptides is performed
offline with strong cation exchange (SCX) resin or reversed-phase liquid 2.4. Identification of metabolites in single species PDEV
chromatogram (RPLC) to improve the identification comprehensiveness
of proteins [53]. As we know, in-gel methods have some important Plants are a rich source of highly diverse metabolites with important
advantages including differentiating splice isoforms, degrading pharmacological properties. Therefore, in addition to lipids, proteins,

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and nucleic acids, PDEVs are enriched with secondary metabolites ac­ an activation effect on the Wnt/Tcf4 signaling pathway, promoting the
cording to the species plant they are derived from [44]. This is the growth of intestinal stem cells [67]. Lipids in ginger exosome-like
largest compositional difference between PDEVs and mammalian exo­ nanoparticles (GELNs), rather than RNAs or proteins, are responsible
somes. These metabolites are small molecular bioactive compounds for the inhibition of the nucleotide-binding domain and leucine-rich
which can be divided into two classes: hydrophilic metabolites and lipid repeat-containing family, pyrin domain-containing 3 (NLRP3) inflam­
metabolites. Currently, methods for the identification of metabolites masome activation [68]. Almost all species PDEVs contain PA, which
include HPLC, LC/gas chromatography (GC)-MS, and nuclear magnetic plays an important role in maintaining the duration and amount of
resonance (NMR) [51,60–63]. Among them, HPLC is generally used for PDEVs accumulation in the gut. PA is also involved in multiple biolog­
the detection of single metabolite, such as naringin, 6-gingerol, or 6-sho­ ical functions, including cell proliferation, transformation, and differ­
gaol [51,63]. LC/GC-MS or NMR-based metabolomics is a novel tech­ entiation [67]. Zhang’s group found that PA lipids play a critical role in
nique to characterize the distinct metabolite profiles of single species mediating the absorption of EVs by specific bacteria. PA is required for
PDEV, and LC/GC-MS is the most widely used technology at present. GELNs uptake by gut bacteria Lactobacillus rhamnosus (LGG). Depletion
Before analysis, metabolites of single species PDEV should be isolated of GELN PA lipids leads to a significant reduction in GELN-positive LGG.
and extracted. For lipid metabolites, the extraction method is the same Whereas addition of PA to PA-depleted GELNs rescues the uptake of
as that of above mentioned for lipids. For hydrophilic metabolites, GELNs by LGG [24]. Liu et al. found that Arabidopsis EVs contained a
PDEVs and internal standard are dissolved in methanol : water : chlo­ much higher level of PA than its leaf tissue, and they might be the
roform (2.5 : 1: 1, v/v/v). After centrifugation, the supernatant is precursors for the biosynthesis of plant hormone jasmonate during
collected. Then methanol : chloroform (1 : 1, v/v) was added to the wound-triggered self-healing [69]. In addition, in the presence of cal­
pellet and centrifuged. The two supernatants are combined and mixed cium, PA is highly fusogenic and is thought to induce inter-vesicular
with water. After centrifuged at 16,000 g for 30 min, the upper aqueous fusion [70]. GELNs can be selectively taken up by the periodontal
phase is collected and dried in a vacuum drier [61]. Hydrophilic inter­ pathogen Porphyromonas gingivalis via the interaction between PA and
action LC with positive-ion mode MS is used to separate polar metabo­ hemin-binding protein 35 (HBP35) on the surface of P. gingivalis. It is
lites. While C18 chromatography with negative-ion mode MS and C8 worth noting that only unsaturated PA could mediate GELNs to interact
chromatography with positive-ion mode MS are used to profile metab­ with HBP35 [71]. PC can enhance the migration of PDEVs from intestine
olites of intermediate polarity and lipids, respectively [64]. For each of to liver. Teng et al. found that PC-enriched grapefruit EVs were prefer­
the methods, metabolite identities are confirmed using authentic refer­ entially taken up by Ruminococcaceae sp. (TSD-27) [24].
ence standards or reference samples. It is worth noting that samples Ether-phospholipids are one distinct type of phospholipids in which the
should be derivatized before GC-MS analysis. The identification of each sn-1 arm of the glycerol backbone is linked by an ether bond. It was
metabolite is conducted using a data system from different companies, worth noting that Catharanthus roseus-derived EVs contained over 36.5%
such as Thermo Scientific Chromeleon Chromatography Data System ether-phospholipids, which may be responsible for the strongly immu­
(CDS) [61]. In general, LC/GC-MS is a highly sensitive and noregulatory effect of Catharanthus roseus-derived EVs [72]. Lipidomic
high-throughput analytical technique for targeted or untargeted studies analysis showed that 69.8% of total lipids in ginseng EVs were glyco­
of metabolites in single species PDEV. lipids, and diacylglycerol was one of the most varied lipids in ginseng
EVs and their plant extracts [45]. Ginseng EVs exhibit anti-senescence
3. Function analyses of different chemical components in single and anti-pigmentation effects in senescent human primary cells. This
species PDEV may be partially attributed to that vesicular diacylglycerol activate the
protein kinase C (PKC) signaling pathway, leading to an increased
3.1. Lipids expression of HMGB1 in the cells. Thus, diacylglycerol may be a po­
tential ginseng EVs’ glycolipid biomarker which could mediate inter­
Phospholipids, sterols, and sphingolipids are three major lipid classes cellular communication between plant and human cells [45]. Cao et al.
in plant lipidomes. However, comparative lipid profiles of different believed that the main glycolipids in ginseng EVs were dicgalactose
species PDEVs illustrate that the lipid class enriched in PDEVs are monoacyl glycerol (59.4%) and ceramide (13.8%), which were simple
phospholipids including phosphatidic acid (PA), phosphatidylethanol­ sphingolipids and had not been detected in other species plant-derived
amine (PE), phosphatidylglycerol (PG), and phosphatidylcholine (PC), EVs [73]. They found that this type of ginseng EVs significantly pro­
etc. The total lipid classes and composition in single species PDEV may moted the polarization of M2 macrophages toward their M1 phenotype,
differ from those of their parental plant extracts. For example, the total sustained M1 polarization, and maintained the production of cytokines
lipid classes of ginseng PDEVs are significantly higher than those of and cytotoxic hydroxyl radicals, leading to increased apoptosis of cancer
ginseng extracts, while the lipid classes of Arabidopsis leaf PDEVs are cells. Considering that ceramides are identified as one type of Toll-like
much lower than those of its plant extract [45]. Although the type of receptor (TLR4) agonists and have been demonstrated to be powerful
fatty acids identified in the celery root lysate and celery root-derived EVs tumor suppressors [74,75], they concluded that ceramide lipids of
are largely similar, the proportions of these fatty acids varied [65]. ginseng EVs might play an important role in macrophage polarization
Moreover, lipid compositions of PDEVs from different species plants are via TLR4 activation [73]. The high content (46%) of sphingolipid gly­
also different. For example, lipids in ginger- and turmeric-derived EVs cosylinositolphosphoceramides in the lipidomic profile of Arabidopsis
are mainly PAs (35.2% and 34.4%, respectively), whereas PAs in leaf EVs promote the secretion of EVs and reactive oxygen species (ROS)
grapefruit and orange juice are only 3.5% and 5% of the total lipids, burst in plants, providing valuable insights into the biogenesis and
respectively [60]. In contrast, the majority of the lipid in grapefruit or function of plant EVs [76]. There are different types of fatty acids (e.g.,
orange juice is PC (36.2% and 25%, respectively), and PE in orange palmitic acid 16:0, palmitoleic acid 16:1, and linoleic acid 18:2)
juice-derived EVs is about 40% [60]. Meanwhile, the major lipid com­ detected in celery root derived-EVs. These fatty acids have
ponents of tea leaf-derived EVs are PA (32.7–45.7%), PG (19.3–22.7%), anti-inflammatory properties and enable their corresponding PDEVs to
PC (13.6–19.2%), and phosphatidylinositol (PI, 5.9–6.2%) [66]. regulate (or suppress) the immune response [65]. In general terms,
Therefore, lipid classes in single species PDEV and corresponding plant different species PDEVs contain several classes of lipids, such as phos­
extracts are inconsistent, and lipid composition varies with PDEVs from pholipids, glycolipids, sphingolipids, and fatty acids. It is also note­
different species plants. worthy that PDEVs of all species investigated so far were found to be
Lipids play important roles in PDEV stability, uptake, and other devoid of cholesterol [24,51,77]. Several studies suggest that lipids in
biological functions. Zhang’s group found that the co-assembly of the PDEVs can not only regulate the uptake of PDEVs by cells or bacteria,
liposome-like nanoparticles with the lipids from grape-derived EVs had but also have a variety of biological functions by regulating different

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signaling pathways (Wnt/Tcf4, PKC, NLRP3, toll-like receptor-4, etc.) in EVs contained heat shock protein family A member 8 (HSPA8). And
recipient cells. And a thorough understanding of the component of each the EVs could be delivered to target intestinal epithelial cells and pre­
lipid, as well as its action mechanism, will be beneficial in the devel­ vent colitis in a mouse model. This is mainly due to the selective binding
opment of effective single species PDEV-based therapeutic strategies. of intestinal aryl hydrocarbon receptor (AhR) with HSPA8 on the EVs
which subsequent activates the receptor-signaling pathway, leading to
3.2. Proteins the induction of Constitutive Photomorphogenic Homolog Subunit 8
(COPS8) and the generation of a series of anti-microbial peptides [86].
Many functional proteins in mammalian exosomes have been iden­ Approximately 26% of the proteins in Arabidopsis EVs have been iden­
tified to be involved in basic metabolic processes, proteolysis, vesicular tified to be associated with biotic and abiotic stress responsibility.
transport, and cytoskeleton formation of their parent cells [78,79]. RPM1-interaction protein was highly induced in the EVs in response to
However, the relationship between functional proteins in single species stress and/or immunity of Arabidopsis [87]. 237 proteins were unam­
PDEV and their roles in human health is less explored. Proteins in PDEVs biguously identified in EVs from sunflower seedlings. Among them,
can be roughly divided into membrane proteins and cytosolic proteins. PMR5 and Gnk2 antifungal proteins could bind to fungal pathogens and
Membrane proteins are important structural components of the vesicle inhibit seedling growth. Interestingly, a comparative analysis revealed
membrane and play active roles in cell-cell or interspecies communi­ that 24 protein families detected in sunflower EVs were also identified in
cation. Meanwhile, most membrane proteins are transmembrane pro­ EVs isolated from Arabidopsis leaves [88]. Pocsfalvi et al. revealed that
teins, which span the lipid bilayer and are partially exposed on both tomato PDEVs containing 14-3-3 proteins, endoplasmic reticulum
sides of the membrane to serve as transporters, channels, anchorage luminal binding proteins and GTP binding proteins had a significant
sites, or signal transductors. Pocsfalvi et al. studied the proteins in anti-inflammatory effect [89]. Besides, a well-known mammalian EVs
PDEVs from several citrus species [25,52,80]. Each sample contained marker, Apoptosis-Linked gene-2 Interacting protein X (ALIX), was also
approximately 600–800 proteins, 440 of which were co-expressed in all found in EVs from germinated kiwi pollen (Actinidia chinensis Planch.)
four species (C. sinensis, C. limon, C. paradisi, and C. aurantium). 87% of [90]. In general, the data of proteins in PDEVs are very limited. To date,
these proteins were aligned to EVPedia (a proteins database of CD63, CD81, and CD9 have been identified as possible markers of
mammalian EVs) [25]. Patellin-3-like protein (PTL3) and clathrin, mammalian exosomes while markers of PDEVs have not been clearly
which are related to vesicle formation and transport, were highly reported yet. Thus, there is a need of considerable research for identi­
expressed in all citrus samples. PTL3 is a plasma-membrane protein and fication of a broad range of different species PDEVs’ proteins, as well as
was also found in other species PDEVs, such as Arabidopsis, zucchini, and revealing of their roles in biological and pharmacological activities.
Glycine max [81]. Through binding to hydrophobic molecules such as In terms of the biological functions of proteins, it is necessary to
phosphoinositol, PTL3 is involved in vesicle and membrane transport mention post-translational modifications (PTMs). By covalently binding
and promotes the transfer of PDEVs between different cellular com­ the chemical groups of small molecules to their amino acid side chains,
partments. Clathrin is a well-known scaffold protein that plays a major the physical or chemical properties, conformations, binding abilities, or
role in the formation of clathrin-coated vesicles [25]. Another mem­ functions of proteins can be directly changed [91]. The same protein
brane protein, aquaporin, has also been detected in EVs from broccoli could harbor various PTMs, which might exhibit different activities or
plant and is reported to play an important role in vesicle stability [82]. functions [92]. PTMs are not only an important way to regulate protein
PDEVs from C. clementina contain 1018 proteins, 162 of which are functions, but also considered as a switch box for cell signaling [93].
transport-related, including 71 transmembrane transporters, 53 Indeed, there have been many studies on PTM proteins in mammalian
vesicle-mediated transporters, and 50 intracellular transporters [80]. EVs. Emerging evidences suggest that PTMs play a vital role in various
Song et al. found that mannose-specific binding protein II lectin was biological functions of EVs, such as EV biogenesis, sorting, and cellular
highly expressed on the surface of garlic-derived EVs and could specif­ recognition [94–96]. Phosphorylated, glycosylated, ubiquitinated,
ically interact with CD98 receptors on HepG2 cells to trigger the inter­ SUMOylated, oxidized, or palmitoylated proteins within the EVs have
nalization of garlic-derived EVs [83]. This allowed garlic-derived EVs to been studied with proteomic or biochemical analyses [93,97]. For
exhibit anti-inflammatory effect in vitro by down-regulating the proin­ plants, PTMs provide a faster reaction mechanism than protein synthesis
flammatory factors of HepG2 cells. Similarly, after the ginseng-derived to cope with environmental stress [98]. Remarkably, proteomic studies
EVs were treated with protease K, their uptake by M2-like macro­ in plants suggest that the number of proteins with PTMs is significantly
phages was significantly reduced, demonstrating the important role of higher [99–103], while PTMs have not been mentioned in the studies of
the proteins in PDEV uptake pathway [73]. PDEVs. Several reasons limit the large-scale analysis of protein PTMs in
Except some membrane proteins, such as membrane channel/ PDEVs: (1) low-abundance PTM proteins; (2) interference from proteins
transporters (e.g., aquaporin and chloride channels), most of the pro­ and metabolites in PDEVs; (3) function of different PTMs and the
teins detected in ginger-derived EVs are cytosolic proteins such as actin regulation on their target proteins’ function [98,104]. Discovery of these
and proteolysis enzymes [51]. PDEVs isolated from fruits and vegetables mysteries will be very helpful in improving the understanding of the
have strong antioxidant capacity, which is mainly related to the anti­ biological functions of PDEVs and exploiting PDEVs from different
oxidant components of the plants. And the antioxidants can be divided perspectives.
into enzymatic antioxidants and non-enzymatic antioxidants [25,59].
Several different hydrolases are also highly presented in PDEVs, such as 3.3. Nucleic acids
ATPase, pectinesterase, phospholipase, amylase, β-galactosidase, and
adenosine homocysteine hydrolase [25]. Other oxidoreductases such as Besides proteins, many nucleic acids such as DNA, siRNA, and
catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), ascor­ miRNA are also identified in PDEVs. PDEVs could keep the stability of
bate peroxidase (APX), and glutathione reductase (GPX) are also highly the miRNAs in the digestive tract and play regulatory functions on
presented in these PDEVs and protect cells from oxidative damage by mammalian bodies [105,106]. Growing evidences indicated that miR­
scavenging the ROS in the cells [25,84]. Furthermore, Hu et al. found NAs of PDEVs can target mammalian genes and thus participate in
that Taraxacum officinale-derived EVs enriched a variety of functional cell-to-cell communication among the organisms of different species.
proteins, which showed oxidoreductase activity and participated in the Small RNAs (sRNAs) are 21–24 nucleotide (nt) non-coding signaling
metabolic and oxoacid metabolic processes of carboxylic acid. Tarax­ molecules that can travel between cells and even between hosts and
acum officinale-derived EVs could mediate the butyrate level in rats, pathogens, regulating a variety of biological functions by controlling the
reducing their intermittent hypoxia-induced hypertension [85]. In translation of target mRNAs. For example, sRNAs in Arabidopsis EVs can
addition to enzymes, Zhang’s group found that mulberry bark-derived accumulate at the infection sites and be taken up by fungal pathogen

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S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

Botrytis cinerea [21]. The transferred host sRNAs induce the silencing of application for inflammatory bowel disease prevention. Furthermore,
pathogenicity-critical fungal genes to achieve PDEVs RNA-based im­ Zhang’s group found that aly-miR396a-5p- and rlcv-miRrL1-28-3p-in
mune responses. ginger-derived EVs could target Nsp12 genes of the lung epithelial
sRNAs in plants can be divided into two major classes: miRNAs and cells that had been exposed to severe acute respiratory syndrome
small interfering RNAs (siRNAs). Compared with siRNAs, miRNAs are coronavirus 2 (SARS-CoV-2), prevent the apoptosis of the cells, and
the most differentially represented sRNAs in PDEVs [107]. To better thereby inhibit lung inflammation [112]. In addition to ginger, miRNAs
understand the expression and function of miRNAs in different species in PDEVs from soybean, Hami melon, grapefruit, tomato, or pear can
PDEVs, Xiao et al. isolated PDEVs from 11 edible fruits and vegetables (i. also target different regions within SARS-CoV-2. After oral or intranasal
e., blueberry, coconut, ginger, grapefruit, Hami melon, kiwifruit, or­ administration, these PDEVs could accumulate in lung tissues and treat
ange, pea, pear, soybean, and tomato) [2]. They identified 32 to 127 coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 [116].
miRNAs per species from 11 samples, of which the highly expressed Besides, Yu et al. verified that EVs from Rehmanniae Radix, a well-known
miRNAs were closely associated with inflammatory response and traditional Chinese herb, could alleviate LPS-induced lung inflamma­
cancer-related pathways. 24 miRNAs were identified in bitter melon tion. The main mechanism is that miR-7972 in R. Radix-derived EVs
EVs, of which 11 miRNAs mediated the anti-inflammatory activity of targeted the G protein-coupled receptor 161 (GPR161), activated the
target cells by regulating the expression of NLRP3 mRNA [26]. Bitter Hedgehog pathway, and decreased the production of pro-inflammatory
melon-derived EVs could reduce the resistance of oral squamous cell cytokines (IL-1β, IL-6, and TNF-α) [117]. Therefore, most of the
carcinoma to 5-Fluorouracil and enhance the antitumor efficacy of the anti-inflammatory studies of PDEVs focus on colitis and pneumonia, and
drug by down-regulating NLRP3 in the cells [108]. Potesta et al. also the research needs to be further extended to other inflammations.
found that miRNAs in PDEVs from Moringa oleifera seeds could target In addition to the widely reported anti-inflammatory and antitumor
human apoptosis-related genes, promote apoptosis of the tumor cells, effects, miRNAs of PDEVs also have excellent efficiency in stimulating
and thereby reduce tumor cell viability [109]. Similarly, Zhu et al. found the neural differentiation of stem cells and anti-photoaging. For
that miR167a enriched in broccoli EVs could target IRS1, which is a key example, miRNAs of ginseng could be internalized by mammalian stem
gene involved in the phosphatidylinositol 3 kinase (PI3K) signaling cells and promote neural recovery by up-regulating the PI3K signaling
pathway, thus inducing apoptosis of human pancreatic cancer cells. pathway. Specifically, 19 genes of stem cells including Tmem100, Vrk1,
Notably, antitumor effect of broccoli EVs was enhanced by selenium and LOC103689968 which relate to neural differentiation, maturation,
biofortification [110]. Besides, 15 mature miRNAs in coffee-derived EVs and functionalization were up-regulated by the miRNAs [56].
were identified by whole-EV RNA sequencing. And two target genes Momordica Charania-derived EVs could attenuate
(KMT2C and ZNF773) which specifically associate with the miRNAs ischemia-reperfusion-induced damage to the blood brain barrier and
were selected using a MapReduce-based MicroRNA Target Prediction inhibit neuronal apoptosis probably via their miRNA5266 [118]. Taken
Method. Coffee-derived EVs significantly suppressed the proliferation of together, compared with lipids and proteins, sRNAs of PDEVs exhibit
hepatocellular carcinoma cells, indicating their therapeutic effects on more biological activities on targeting cells including
chronic liver diseases [111]. These studies indicated that miRNAs in anti-inflammatory, anti-tumor, antiviral, and nerve differentiation pro­
different species PDEVs play a potential role in counteracting motion activities. However, most studies just focused on the functional
tumorigenesis. studies of miRNAs in different species PDEVs except ginger, their spe­
Moreover, PDEVs from ginger have a good anti-inflammatory effect, cific molecular mechanisms are still unclear. There are few studies on
which is mainly attributed to their miRNAs [24,51,112]. A total of 116 DNA in PDEVs and only one case has been found so far (i.e., mito­
miRNAs were identified in ginger derived-EVs. The expression of 27 chondrial DNA, mtDNA). As a major effector molecule, mtDNA could be
miRNAs in PDEVs was higher than that in its ginger extract, and three internalized into tumor-associated macrophages (TAMs) with Artemisia
novel miRNAs (novel 18, novel 20, and novel 27) were specifically annua PDEVs, initiate the cGAS-STING pathway, reprogram TAMs, thus
expressed in ginger-derived EVs. These miRNAs were mainly involved in boosting anti-tumor immunity and inhibiting tumor growth in a mouse
immunomodulatory or metabolic regulation. After being specifically model of lung cancer [119]. In addition to miRNA, plant-derived DNA
internalized by intestine cells including intestinal epithelial cells (IECs) also exhibits a wide range of biological activities. Thus, DNAs in PDEVs
and macrophages, ginger-derived EVs could counteract lipopolysac­ and their molecular mechanisms should be further addressed.
charide (LPS)-induced inflammation by reducing the expression of
proinflammatory cytokines (TNF-α, IL-6 and IL-1β) and increasing the 3.4. Metabolites
expression of anti-inflammatory cytokines (IL-10 and IL-22) in macro­
phages through their miRNAs [51,113]. Notably, Zhang’s group found Different species PDEVs are widely reported to have various bene­
that ginger PDEVs could be taken up not only by mammalian cells, but ficial effects on human health and disease [120–124]. The most abun­
also by gut microbiota. Ginger derived-EVs could be preferentially taken dant components in fruits and vegetables are antioxidants, which relate
up by LGG due to their lipid composition. Mdo-miR7267–3p in to a risk reduction in a series of human diseases. Similarly, PDEVs have
ginger-derived EVs specifically acted on LGG mono-oxygenase ycnE, high content of total antioxidants. EVs derived from golden cherry
thus increasing the generation of IL-22 and thereby enhancing intestinal (Physalis minima) have potential antioxidant activity to mitigate the risk
repair. This observation revealed an important molecular mechanism of photoaging [125]. Interestingly, Logozzi, M. et al. found that PDEVs
that how the miRNAs in PDEVs cross-talk with gut microbiota to from organic agriculture showed a higher antioxidant level compared to
maintain gut health [24]. In addition to ginger, 127 known miRNAs those from conventional agriculture [84]. In addition to the enzymatic
have been found in PDEVs derived from garlic. Among them, 23 specific antioxidants mentioned in the protein section above, there is also a class
miRNAs, especially han-miR3630–5p with a relatively higher abun­ of non-enzymatic antioxidants, such as ascorbic acid (vitamin C) and
dance, could directly target human gene encoding factors of inflam­ glutathione. The activities of unstable ascorbic acid are well protected
mation and inhibited the expression of TLR4. Intake of garlic-derived by the membranes of PDEVs. Vitamin C-containing PDEVs from Citrus
EVs also altered the gut microbiota profile of colitis mice by recovering limon L. juice and strawberry juice could be uptake by mesenchymal
the relative abundance of Lachnospiraceae and reducing the relative stromal cells (MSC) and had a significant protective effect against
abundance of Helicobacter [114]. Besides, miR-396e, another abundant oxidative stress [126,127]. However, according to the results of
miRNA of garlic PDEVs, is the key component to regulate PFKFB3 metabolomics, Berger, E. et al. found that PDEVs from orange juice
expression, which could inhibit inflammatory response and enhance (ONVs) did not contain vitamin C, but contained carbohydrates
lipid metabolism in hepatocytes in high-fat diet-fed mice [115]. These (glucose, fructose, sucrose) and amino acids (alanine, asparagine
studies indicated that EVs derived from ginger or garlic had a potential isoleucine, threonine, leucine). The concentrated amino acids and

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S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

Fig. 3. Overview of target cells and biological functions of PDEVs from different species plant sources. Pictures of ‘PDEVs source’ are from the internet.

bioactive lipids enable ONVs to reverse diet-induced gut modifications TLR4/TRIF-dependent manner. This enables ginger-derived EVs to
and treat obesity-associated intestinal complications [60]. PDEVs from delay the development of alcohol-induced liver damage [62].
another citrus fruit, grapefruit, exhibit high amounts of organic acids Turmeric-derived EVs containing high levels of curcumin, lipids, and
(mainly glycolic and citric acids) and amino acids (mainly leucine/iso­ proteins showed excellent anti-inflammatory and antioxidant properties
leucine and aspartic acid). All these metabolites delivered by grapefruit via regulating the expressions of the pro-inflammatory cytokines and
vesicles may be absorbed by melanoma cells and exhibit anticancer antioxidant genes, or mediating inactivation of the nuclear factor
properties [61]. PDEVs also contain large amounts of polyphenols and kappa-B (NF-κB) pathway [133]. Therefore, PDEVs from different spe­
flavonoids. For example, tea leaf-derived EVs contained epi­ cies plant are also excellent carriers of secondary metabolites (hydro­
gallocatechin gallate, vitexin-2-O-rhamnoside, vitexin, myricetin-3-O-­ philic metabolites and lipid metabolites). Compared with those in plant
rhamnoside, kaempferol-3-O-galactoside, and myricetin. These active extracts, the metabolites in PDEVs exhibit better functionalities due to
small molecules together with proteins and lipids enable tea leaf-derived the protection of their PDEVs.
EVs to induce the apoptosis of tumor cells [128]. Cannabis-derived EVs
containing high level cannabidiol significantly induced hepatocellular 4. Conclusion and perspective
carcinoma cell death by activating mitochondrial-dependent apoptosis
signaling pathways in the cells, while they had no significant inhibitory In recent years, with the explosive development of mammalian
effect on normal cell growth [129]. Notably, Eom, J. Y. et al. found that exosomes, PDEVs with similar structure have gradually attracted the
EVs isolated from different cannabis parts (root, seed, hemp sprout, or attention of researchers. Increasing evidences suggest that PDEVs are
leaf) exhibited similar biological functions [130]. Puerarin, another potential functional vesicles with multiple health benefits, which are
flavonoid, is enrich in Pueraria lobata-derived EVs. Wu, J. et al. found endowed by their unique compositions. In this review, we focus on the
that Pueraria lobata-derived EVs could drive M1 macrophages toward different components of single species PDEV including lipids, proteins,
their M2 phenotypes and therefore enhance the anti-inflammatory ef­ nucleic acid, and metabolites, and discuss their identification methods
fects of the macrophages [131]. In addition to those several miRNAs and healthy functions. Due to the complexity of PDEVs, different mass-
mentioned in the nucleic acids section above, ginsenosides, another based omics methods (e.g., lipidomics, proteomics, and metabolomics)
important active component of ginseng, has been found enriched in and the next generation sequencing technology are commonly used to
ginseng-derived EVs. Rb1 (64.5%) and Rg1 (31.1%) are the main type of achieve the comprehensive identifications of different components in
ginsenosides encapsulated in EVs. They could downregulate the IκBα, single species PDEV. Lipid compositions and membrane proteins of
JNK, and ERK signaling pathways as well as the c-Fos, c-Jun, and PDEVs allow them to target different cells, bacteria, or tissues. Through
NFATc1 genes of marrow-derived macrophages and strongly hindered their components (especially miRNAs and metabolites), internalized
osteoclast differentiation [132]. In general, PDEVs contain a large PDEVs exhibit different biological activities such as immune-regulation,
number of plant-derived small molecular metabolites, which in turn inflammation modulation, tissue regeneration, antitumor effect, etc.
endow the vesicles with strong antioxidant capacity. At the same time, (Fig. 3). Besides, PDEVs have several merits including large-scale pro­
together with the proteins and lipids in the vesicles, PDEVs exert duction, high biocompatibility, low cost, and drug delivery capability.
anti-inflammatory and anti-tumor effects. These combined features highlight the great potential of different spe­
Besides being encapsulated inside PDEVs, when they are lipophilic cies PDEVs in future therapeutics.
enough, metabolites can also be enriched in the membranes of PDEVs Although great progress has been made in the field of PDEVs in
[57]. For example, naringin and its metabolite naringenin were detected recent decade, there are still some challenges remaining ahead of the
in the membrane of grapefruit EVs [63]. Besides, sulforaphane, which is clinical applications of PDEVs. First, the extraction method for single
highly enriched in the membranes of broccoli-derived EVs, plays a role species PDEV is simple. The most commonly used method is density
in the prevention of mouse colitis through adenosine gradient centrifugation while optimization of the extraction process is
monophosphate-activated protein kinase (AMPK) activation [13]. rarely studied. Second, the lack of markers, especially surface proteins,
Similarly, large amounts of 6-gingerol and 6-shogaol in ginger-derived limits the differentiation of PDEVs from different species plants. More­
EVs would participate in a great part of the reduction of acute colitis, over, most studies do not systematically study the composition and
enhancement of intestinal repair, and prevention of colitis-associated function of PDEVs. Some only focus on the characterizations of the
cancer [51]. Moreover, shogaol plays an important role in the induc­ components in PDEVs, while others only study the functions of PDEVs
tion of nuclear factor erythroid 2-related factor 2 (Nrf2) in a [52,107,134]. The relationships between the active components in

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S. Zhou et al. Trends in Analytical Chemistry 167 (2023) 117274

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