2023 Volume 3
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Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects

  • These authors contributed equally: Shanghui Gao

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  • In recent decades, natural products have gained increasing attention as potential therapies for chronic diseases, including inflammation and cancer. Among them, a hot water extract derived from seven traditional Chinese herbs (Lycii fructus, Crataegi fructus, Phyllanthi fructus, Chrysanthemi flos, Coicis semen, Ganoderma lucidum, and Zizyphi fructus), referred to as LLA, has demonstrated potent antioxidative effects. In line with this, our study aimed to investigate the anti-inflammatory properties of LLA and explore its potential for cancer prevention. To evaluate the anti-inflammatory effects of LLA, we employed a dextran sulfate sodium (DSS)-induced mouse colitis model. Daily oral administration of LLA significantly suppressed colitis symptoms, improving the disease activity index and mitigating colon tissue damage. Moreover, LLA treatment effectively inhibited the production of proinflammatory cytokines, including IL-6, TNF-α, and MCP-1. In addition to its anti-inflammatory effects, LLA exhibited remarkable efficacy in suppressing mouse colon carcinogenesis induced by azoxymethane (AOM) and DSS. Administration of LLA during the AOM/DSS treatment resulted in a significant reduction in tumor occurrence and growth, highlighting its potential as a preventive agent against colon cancer. Furthermore, we investigated the antitumor effect of LLA using a mouse sarcoma S180 solid tumor model. Although LLA did not demonstrate significant antitumor effects on established tumors, its application prior to tumor formation significantly inhibited tumor occurrence and growth. Collectively, these findings underscore the beneficial effects of LLA administration in the context of inflammatory diseases and highlight its potential as a preventive measure against cancer.
  • Aquaporins (AQPs) constitute a large family of transmembrane channel proteins that function as regulators of intracellular and intercellular water flow[1,2]. Since their first discovery in the 1990s, AQPs have been found not only in three domains of life, i.e., bacteria, eukaryotes, and archaea, but also in viruses[3,4]. Each AQP monomer is composed of an internal repeat of three transmembrane helices (i.e., TM1–TM6) as well as two half helixes that are formed by loop B (LB) and LE through dipping into the membrane[5]. The dual Asn-Pro-Ala (NPA) motifs that are located at the N-terminus of two half helixes act as a size barrier of the pore via creating an electrostatic repulsion of protons, whereas the so-called aromatic/arginine (ar/R) selectivity filter (i.e., H2, H5, LE1, and LE2) determines the substrate specificity by rendering the pore constriction site diverse in both size and hydrophobicity[59]. Based on sequence similarity, AQPs in higher plants could be divided into five subfamilies, i.e., plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), NOD26-like intrinsic protein (NIP), X intrinsic protein (XIP), and small basic intrinsic protein (SIP)[1017]. Among them, PIPs, which are typically localized in the cell membrane, are most conserved and play a central role in controlling plant water status[12,1822]. Among two phylogenetic groups present in the PIP subfamily, PIP1 possesses a relatively longer N-terminus and PIP2 features an extended C-terminus with one or more conserved S residues for phosphorylation modification[5,15,17].

    Tigernut (Cyperus esculentus L.), which belongs to the Cyperaceae family within Poales, is a novel and promising herbaceous C4 oil crop with wide adaptability, large biomass, and short life period[2327]. Tigernut is a unique species accumulating up to 35% oil in the underground tubers[2830], which are developed from stolons and the process includes three main stages, i.e., initiation, swelling, and maturation[3133]. Water is essential for tuber development and tuber moisture content maintains a relatively high level of approximately 85% until maturation when a significant drop to about 45% is observed[28,32]. Thereby, uncovering the mechanism of tuber water balance is of particular interest. Despite crucial roles of PIPs in the cell water balance, to date, their characterization in tigernut is still in the infancy[21]. The recently available genome and transcriptome datasets[31,33,34] provide an opportunity to address this issue.

    In this study, a global characterization of PIP genes was conducted in tigernut, including gene localizations, gene structures, sequence characteristics, and evolutionary patterns. Moreover, the correlation of CePIP mRNA/protein abundance with water content during tuber development as well as subcellular localizations were also investigated, which facilitated further elucidating the water balance mechanism in this special species.

    PIP genes reported in Arabidopsis (Arabidopsis thaliana)[10] and rice (Oryza sativa)[11] were respectively obtained from TAIR11 (www.arabidopsis.org) and RGAP7 (http://rice.uga.edu), and detailed information is shown in Supplemental Table S1. Their protein sequences were used as queries for tBLASTn[35] (E-value, 1e–10) search of the full-length tigernut transcriptome and genome sequences that were accessed from CNGBdb (https://db.cngb.org/search/assembly/CNA0051961)[31,34]. RNA sequencing (RNA-seq) reads that are available in NCBI (www.ncbi.nlm.nih.gov/sra) were also adopted for gene structure revision as described before[13], and presence of the conserved MIP (major intrinsic protein, Pfam accession number PF00230) domain in candidates was confirmed using MOTIF Search (www.genome.jp/tools/motif). To uncover the origin and evolution of CePIP genes, a similar approach was also employed to identify homologs from representative plant species, i.e., Carex cristatella (v1, Cyperaceae)[36], Rhynchospora breviuscula (v1, Cyperaceae)[37], and Juncus effusus (v1, Juncaceae)[37], whose genome sequences were accessed from NCBI (www.ncbi.nlm.nih.gov). Gene structure of candidates were displayed using GSDS 2.0 (http://gsds.gao-lab.org), whereas physiochemical parameters of deduced proteins were calculated using ProtParam (http://web.expasy.org/protparam). Subcellular localization prediction was conducted using WoLF PSORT (www.genscript.com/wolf-psort.html).

    Nucleotide and protein multiple sequence alignments were respectively conducted using ClustalW and MUSCLE implemented in MEGA6[38] with default parameters, and phylogenetic tree construction was carried out using MEGA6 with the maximum likelihood method and bootstrap of 1,000 replicates. Systematic names of PIP genes were assigned with two italic letters denoting the source organism and a progressive number based on sequence similarity. Conserved motifs were identified using MEME Suite 5.5.3 (https://meme-suite.org/tools/meme) with optimized parameters as follows: Any number of repetitions, maximum number of 15 motifs, and a width of 6 and 250 residues for each motif. TMs and conserved residues were identified using homology modeling and sequence alignment with the structure resolved spinach (Spinacia oleracea) SoPIP2;1[5].

    Synteny analysis was conducted using TBtools-II[39] as described previously[40], where the parameters were set as E-value of 1e-10 and BLAST hits of 5. Duplication modes were identified using the DupGen_finder pipeline[41], and Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) of duplicate pairs were calculated using codeml in the PAML package[42]. Orthologs between different species were identified using InParanoid[43] and information from synteny analysis, and orthogroups (OGs) were assigned only when they were present in at least two species examined.

    Plant materials used for gene cloning, qRT-PCR analysis, and 4D-parallel reaction monitoring (4D-PRM)-based protein quantification were derived from a tigernut variety Reyan3[31], and plants were grown in a greenhouse as described previously[25]. For expression profiling during leaf development, three representative stages, i.e., young, mature, and senescing, were selected and the chlorophyll content was checked using SPAD-502Plus (Konica Minolta, Shanghai, China) as previously described[44]. Young and senescing leaves are yellow in appearance, and their chlorophyll contents are just half of that of mature leaves that are dark green. For diurnal fluctuation regulation, mature leaves were sampled every 4 h from the onset of light at 8 a.m. For gene regulation during tuber development, fresh tubers at 1, 5, 10, 15, 20, 25, and 35 d after tuber initiation (DAI) were collected as described previously[32]. All samples with three biological replicates were quickly frozen with liquid nitrogen and stored at −80 °C for further use. For subcellular localization analysis, tobacco (Nicotiana benthamiana) plants were grown as previously described[20].

    Tissue-specific expression profiles of CePIP genes were investigated using Illumina RNA-seq samples (150 bp paired-end reads) with three biological replicates for young leaf, mature leaf, sheath of mature leaf, shoot apex, root, rhizome, and three stages of developmental tuber (40, 85, and 120 d after sowing (DAS)), which are under the NCBI accession number of PRJNA703731. Raw sequence reads in the FASTQ format were obtained using fastq-dump, and quality control was performed using fastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc). Read mapping was performed using HISAT2 (v2.2.1, https://daehwankimlab.github.io/hisat2), and relative gene expression level was presented as FPKM (fragments per kilobase of exon per million fragments mapped)[45].

    For qRT-PCR analysis, total RNA extraction and synthesis of the first-strand cDNA were conducted as previously described[24]. Primers used in this study are shown in Supplemental Table S2, where CeUCE2 and CeTIP41[25,33] were employed as two reference genes. PCR reaction in triplicate for each biological sample was carried out using the SYBR-green Mix (Takara) on a Real-time Thermal Cycler Type 5100 (Thermal Fisher Scientific Oy). Relative gene abundance was estimated with the 2−ΔΔCᴛ method and statistical analysis was performed using SPSS Statistics 20 as described previously[13].

    Raw proteomic data for tigernut roots, leaves, freshly harvested, dried, rehydrated for 48 h, and sprouted tubers were downloaded from ProteomeXchange/PRIDE (www.proteomexchange.org, PXD021894, PXD031123, and PXD035931), which were further analyzed using Maxquant (v1.6.15.0, www.maxquant.org). Three dominant members, i.e., CePIP1;1, -2;1, and -2;8, were selected for 4D-PRM quantification analysis, and related unique peptides are shown in Supplemental Table S3. Protein extraction, trypsin digestion, and LC-MS/MS analysis were conducted as described previously[46].

    For subcellular localization analysis, the coding region (CDS) of CePIP1;1, -2;1, and -2;8 were cloned into pNC-Cam1304-SubN via Nimble Cloning as described before[30]. Then, recombinant plasmids were introduced into Agrobacterium tumefaciens GV3101 with the helper plasmid pSoup-P19 and infiltration of 4-week-old tobacco leaves were performed as previously described[20]. For subcellular localization analysis, the plasma membrane marker HbPIP2;3-RFP[22] was co-transformed as a positive control. Fluorescence observation was conducted using confocal laser scanning microscopy imaging (Zeiss LMS880, Germany): The wavelength of laser-1 was set as 730 nm for RFP observation, where the fluorescence was excited at 561 nm; the wavelength of laser-2 was set as 750 nm for EGFP observation, where the fluorescence was excited at 488 nm; and the wavelength of laser-3 was set as 470 nm for chlorophyll autofluorescence observation, where the fluorescence was excited at 633 nm.

    As shown in Table 1, a total of 14 PIP genes were identified from eight tigernut scaffolds (Scfs). The CDS length varies from 831 to 882 bp, putatively encoding 276–293 amino acids (AA) with a molecular weight (MW) of 29.16–31.59 kilodalton (kDa). The theoretical isoelectric point (pI) varies from 7.04 to 9.46, implying that they are all alkaline. The grand average of hydropathicity (GRAVY) is between 0.344 and 0.577, and the aliphatic index (II) ranges from 94.57 to 106.90, which are consistent with the hydrophobic characteristic of AQPs[47]. As expected, like SoPIP2;1, all CePIPs include six TMs, two typical NPA motifs, the invariable ar/R filter F-H-T-R, five conserved Froger's positions Q/M-S-A-F-W, and two highly conserved residues corresponding to H193 and L197 in SoPIP2;1 that were proven to be involved in gating[5,48], though the H→F variation was found in CePIP2;9, -2;10, and -2;11 (Supplemental Fig. S1). Moreover, two S residues, corresponding to S115 and S274 in SoPIP2;1[5], respectively, were also found in the majority of CePIPs (Supplemental Fig. S1), implying their posttranslational regulation by phosphorylation.

    Table 1.  Fourteen PIP genes identified in C. esculentus.
    Gene name Locus Position Intron no. AA MW (kDa) pI GRAVY AI TM MIP
    CePIP1;1 CESC_15147 Scf9:2757378..2759502(–) 3 288 30.76 8.82 0.384 95.28 6 47..276
    CePIP1;2 CESC_04128 Scf4:3806361..3807726(–) 3 291 31.11 8.81 0.344 95.95 6 46..274
    CePIP1;3 CESC_15950 Scf54:5022493..5023820(+) 3 289 31.06 8.80 0.363 94.57 6 49..278
    CePIP2;1 CESC_15350 Scf9:879960..884243(+) 3 288 30.34 8.60 0.529 103.02 6 33..269
    CePIP2;2 CESC_00011 Scf30:4234620..4236549(+) 3 293 31.59 9.27 0.394 101.57 6 35..268
    CePIP2;3 CESC_00010 Scf30:4239406..4241658(+) 3 291 30.88 9.44 0.432 98.97 6 31..266
    CePIP2;4 CESC_05080 Scf46:307799..309544(+) 3 285 30.44 7.04 0.453 100.32 6 28..265
    CePIP2;5 CESC_05079 Scf46:312254..314388(+) 3 286 30.49 7.04 0.512 101.68 6 31..268
    CePIP2;6 CESC_05078 Scf46:316024..317780(+) 3 288 30.65 7.68 0.475 103.06 6 31..268
    CePIP2;7 CESC_05077 Scf46:320439..322184(+) 3 284 30.12 8.55 0.500 100.00 6 29..266
    CePIP2;8 CESC_14470 Scf2:4446409..4448999(+) 3 284 30.37 8.30 0.490 106.90 6 33..263
    CePIP2;9 CESC_02223 Scf1:2543928..2545778(–) 3 283 30.09 9.46 0.533 106.47 6 31..262
    CePIP2;10 CESC_10007 Scf27:1686032..1688010(–) 3 276 29.16 9.23 0.560 106.05 6 26..256
    CePIP2;11 CESC_10009 Scf27:1694196..1696175(–) 3 284 29.71 9.10 0.577 105.49 6 33..263
    AA: amino acid; AI: aliphatic index; GRAVY: grand average of hydropathicity; kDa: kilodalton; MIP: major intrinsic protein; MW: molecular weight; pI: isoelectric point; PIP: plasma membrane intrinsic protein; Scf: scaffold; TM: transmembrane helix.
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    To uncover the evolutionary relationships, an unrooted phylogenetic tree was constructed using the full-length protein sequences of CePIPs together with 11 OsPIPs and 13 AtPIPs. As shown in Fig. 1a, these proteins were clustered into two main groups, corresponding to PIP1 and PIP2 as previously defined[10,49], and each appears to have evolved into several subgroups. Compared with PIP1s, PIP2s possess a relatively shorter N-terminal but an extended C-terminal with one conserved S residue (Supplemental Fig. S1). Interestingly, a high number of gene repeats were detected, most of which seem to be species-specific, i.e., AtPIP1;1/-1;2/-1;3/-1;4/-1;5, AtPIP2;1/-2;2/-2;3/-2;4/-2;5/-2;6, AtPIP2;7/-2;8, OsPIP1;1/-1;2/-1;3, OsPIP2;1/-2;4/-2;5, OsPIP2;2/-2;3, CePIP1;1/-1;2, CePIP2;2/-2;3, CePIP2;4/-2;5/-2;6/-2;7, and CePIP2;9/-2;10/-2;11, reflecting the occurrence of more than one lineage-specific whole-genome duplications (WGDs) after their divergence[50,51]. In Arabidopsis that experienced three WGDs (i.e. γ, β, and α) after the split with the monocot clade[52], AtPIP1;5 in the PIP1 group first gave rise to AtPIP1;1 via the γ WGD shared by all core eudicots[50], which latter resulted in AtPIP1;3, -1;4, and -1;2 via β and α WGDs; AtPIP2;1 in the PIP2 group first gave rise to AtPIP2;6 via the γ WGD, and they latter generated AtPIP2;2, and -2;5 via the α WGD (Supplemental Table S1). In rice, which also experienced three WGDs (i.e. τ, σ, and ρ) after the split with the eudicot clade[51], OsPIP1;2 and -2;3 generated OsPIP1;1 and -2;2 via the Poaceae-specific ρ WGD, respectively. Additionally, tandem, proximal, transposed and dispersed duplications also played a role on the gene expansion in these two species (Supplemental Table S1).

    Figure 1.  Structural and phylogenetic analysis of PIPs in C. esculentus, O. sativa, and A. thaliana. (a) Shown is an unrooted phylogenetic tree resulting from full-length PIPs with MEGA6 (maximum likelihood method and bootstrap of 1,000 replicates), where the distance scale denotes the number of amino acid substitutions per site. (b) Shown are the exon-intron structures. (c) Shown is the distribution of conserved motifs among PIPs, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. (At: A. thaliana; Ce: C. esculentus; PIP: plasma membrane intrinsic protein; Os: O. sativa).

    Analysis of gene structures revealed that all CePIP and AtPIP genes possess three introns and four exons in the CDS, in contrast to the frequent loss of certain introns in rice, including OsPIP1;2, -1;3, -2;1, -2;3, -2;4, -2;5, -2;6, -2;7, and -2;8 (Fig. 1b). The positions of three introns are highly conserved, which are located in sequences encoding LB (three residues before the first NPA), LD (one residue before the conserved L involved in gating), and LE (18 residues after the second NPA), respectively (Supplemental Fig. S1). The intron length of CePIP genes is highly variable, i.e., 109–993 bp, 115–1745 bp, and 95–866 bp for three introns, respectively. By contrast, the exon length is relatively less variable: Exons 2 and 3 are invariable with 296 bp and 141 bp, respectively, whereas Exons 1 and 4 are of 277–343 bp and 93–132 bp, determining the length of N- and C-terminus of PIP1 and PIP2, respectively (Fig. 1b). Correspondingly, their protein structures were shown to be highly conserved, and six (i.e., Motifs 1–6) out of 15 motifs identified are broadly present. Among them, Motif 3, -2, -6, -1, and -4 constitute the conserved MIP domain. In contrast to a single Motif 5 present in most PIP2s, all PIP1s possess two sequential copies of Motif 5, where the first one is located at the extended N-terminal. In CePIP2;3 and OsPIP2;7, Motif 5 is replaced by Motif 13; in CePIP2;2, it is replaced by two copies of Motif 15; and no significant motif was detected in this region of CePIP2;10. PIP1s and PIP2s usually feature Motif 9 and -7 at the C-terminal, respectively, though it is replaced by Motif 12 in CePIP2;6 and OsPIP2;8. PIP2s usually feature Motif 8 at the N-terminal, though it is replaced by Motif 14 in CePIP2;2 and -2;3 or replaced by Motif 11 in CePIP2;10 and -2;11 (Fig. 1c).

    As shown in Fig. 2a, gene localization of CePIPs revealed three gene clusters, i.e., CePIP2;2/-2;3 on Scf30, CePIP2;4/-2;5/-2;6/-2;7 on Scf46, and CePIP2;10/-2;11 on Scf27, which were defined as tandem repeats for their high sequence similarities and neighboring locations. The nucleotide identities of these duplicate pairs vary from 70.5% to 91.2%, and the Ks values range from 0.0971 to 1.2778 (Table 2), implying different time of their birth. According to intra-species synteny analysis, two duplicate pairs, i.e., CePIP1;1/-1;2 and CePIP2;2/-2;4, were shown to be located within syntenic blocks (Fig. 2b) and thus were defined as WGD repeats. Among them, CePIP1;1/-1;2 possess a comparable Ks value to CePIP2;2/-2;3, CePIP1;1/-1;3, and CePIP2;4/-2;8 (1.2522 vs 1.2287–1.2778), whereas CePIP2;2/-2;4 harbor a relatively higher Ks value of 1.5474 (Table 2), implying early origin or fast evolution of the latter. While CePIP1;1/-1;3 and CePIP2;1/-2;8 were characterized as transposed repeats, CePIP2;1/-2;2, CePIP2;9/-2;10, and CePIP2;8/-2;10 were characterized as dispersed repeats (Fig. 2a). The Ks values of three dispersed repeats vary from 0.8591 to 3.0117 (Table 2), implying distinct times of origin.

    Figure 2.  Duplication events of CePIP genes and synteny analysis within and between C. esculentus, O. sativa, and A. thaliana. (a) Duplication events detected in tigernut. Serial numbers are indicated at the top of each scaffold, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (shown in green), transposed (shown in purple), dispersed (shown in gold), and WGD (shown in red). (b) Synteny analysis within and between C. esculentus, O. sativa, and A. thaliana. (c) Synteny analysis within and between C. esculentus, C. cristatella, R. breviuscula, and J. effusus. Shown are PIP-encoding chromosomes/scaffolds and only syntenic blocks that contain PIP genes are marked, i.e., red and purple for intra- and inter-species, respectively. (At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effusus; Mb: megabase; PIP: plasma membrane intrinsic protein; Os: O. sativa; Rb: R. breviuscula; Scf: scaffold; WGD: whole-genome duplication).
    Table 2.  Sequence identity and evolutionary rate of homologous PIP gene pairs identified in C. esculentus. Ks and Ka were calculated using PAML.
    Duplicate 1 Duplicate 2 Identity (%) Ka Ks Ka/Ks
    CePIP1;1 CePIP1;3 78.70 0.0750 1.2287 0.0610
    CePIP1;2 CePIP1;1 77.20 0.0894 1.2522 0.0714
    CePIP2;1 CePIP2;4 74.90 0.0965 1.7009 0.0567
    CePIP2;3 CePIP2;2 70.50 0.1819 1.2778 0.1424
    CePIP2;4 CePIP2;2 66.50 0.2094 1.5474 0.1353
    CePIP2;5 CePIP2;4 87.30 0.0225 0.4948 0.0455
    CePIP2;6 CePIP2;5 84.90 0.0545 0.5820 0.0937
    CePIP2;7 CePIP2;6 78.70 0.0894 1.0269 0.0871
    CePIP2;8 CePIP2;4 72.90 0.1401 1.2641 0.1109
    CePIP2;9 CePIP2;10 76.40 0.1290 0.8591 0.1502
    CePIP2;10 CePIP2;8 64.90 0.2432 3.0117 0.0807
    CePIP2;11 CePIP2;10 91.20 0.0562 0.0971 0.5783
    Ce: C. esculentus; Ka: nonsynonymous substitution rate; Ks: synonymous substitution rate; PIP: plasma membrane intrinsic protein.
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    According to inter-species syntenic analysis, six out of 14 CePIP genes were shown to have syntelogs in rice, including 1:1, 1:2, and 2:2 (i.e. CePIP1;1 vs OsPIP1;3, CePIP1;3 vs OsPIP1;2/-1;1, CePIP2;1 vs OsPIP2;4, CePIP2;2/-2;4 vs OsPIP2;3/-2;2, and CePIP2;8 vs OsPIP2;6), in striking contrast to a single one found in Arabidopsis (i.e. CePIP1;2 vs AtPIP1;2). Correspondingly, only OsPIP1;2 in rice was shown to have syntelogs in Arabidopsis, i.e., AtPIP1;3 and -1;4 (Fig. 2b). These results are consistent with their taxonomic relationships that tigernut and rice are closely related[50,51], and also imply lineage-specific evolution after their divergence.

    As described above, phylogenetic and syntenic analyses showed that the last common ancestor of tigernut and rice is more likely to possess only two PIP1s and three PIP2s. However, it is not clear whether the gene expansion observed in tigernut is species-specific or Cyperaceae-specific. To address this issue, recently available genomes were used to identify PIP subfamily genes from C. cristatella, R. breviuscula, and J. effuses, resulting in 15, 13, and nine members, respectively. Interestingly, in contrast to a high number of tandem repeats found in Cyperaceae species, only one pair of tandem repeats (i.e., JePIP2;3 and -2;4) were identified in J. effusus, a close outgroup species to Cyperaceae in the Juncaceae family[36,37]. According to homologous analysis, a total of 12 orthogroups were identified, where JePIP genes belong to PIP1A (JePIP1;1), PIP1B (JePIP1;2), PIP1C (JePIP1;3), PIP2A (JePIP2;1), PIP2B (JePIP2;2), PIP2F (JePIP2;3 and -2;4), PIP2G (JePIP2;5), and PIP2H (JePIP2;6) (Table 3). Further intra-species syntenic analysis revealed that JePIP1;1/-1;2 and JePIP2;2/-2;3 are located within syntenic blocks, which is consistent with CePIP1;1/-1;2, CePIP2;2/-2;4, CcPIP1;1/-1;2, CcPIP2;3/-2;4, RbPIP1;1/-1;2, and RbPIP2;2/-2;5 (Fig. 2c), implying that PIP1A/PIP1B and PIP2B/PIP2D were derived from WGDs occurred sometime before Cyperaceae-Juncaceae divergence. After the split with Juncaceae, tandem duplications frequently occurred in Cyperaceae, where PIP2B/PIP2C and PIP2D/PIP2E/PIP2F retain in most Cyperaceae plants examined in this study. By contrast, species-specific expansion was also observed, i.e., CePIP2;4/-2;5, CePIP2;10/-2;11, CcPIP1;2/-1;3, CcPIP2;4/-2;5, CcPIP2;8/-2;9, CcPIP2;10/-2;11, RbPIP2;3/-2;4, and RbPIP2;9/-2;10 (Table 3 & Fig. 2c).

    Table 3.  Twelve proposed orthogroups based on comparison of representative plant species.
    Orthogroup C. esculentus C. cristatella R. breviuscula J. effusus O. sativa A. thaliana
    PIP1A CePIP1;1 CcPIP1;1 RbPIP1;1 JePIP1;1 OsPIP1;3 AtPIP1;1, AtPIP1;2,
    AtPIP1;3, AtPIP1;4,
    AtPIP1;5
    PIP1B CePIP1;2 CcPIP1;2, CcPIP1;3 RbPIP1;2 JePIP1;2
    PIP1C CePIP1;3 CcPIP1;4 RbPIP1;3 JePIP1;3 OsPIP1;1, OsPIP1;2
    PIP2A CePIP2;1 CcPIP2;1 RbPIP2;1 JePIP2;1 OsPIP2;1, OsPIP2;4,
    OsPIP2;5
    AtPIP2;1, AtPIP2;2,
    AtPIP2;3, AtPIP2;4,
    AtPIP2;5, AtPIP2;6
    PIP2B CePIP2;2 CcPIP2;2 RbPIP2;2 JePIP2;2 OsPIP2;2, OsPIP2;3
    PIP2C CePIP2;3 CcPIP2;3 RbPIP2;3, RbPIP2;4
    PIP2D CePIP2;4, CePIP2;5 CcPIP2;4, CcPIP2;5 RbPIP2;5
    PIP2E CePIP2;5 CcPIP2;5 RbPIP2;6
    PIP2F CePIP2;6 CcPIP2;6
    PIP2G CePIP2;7 CcPIP2;7 RbPIP2;7 JePIP2;3, JePIP2;4
    PIP2H CePIP2;8 CcPIP2;8, CcPIP2;9 RbPIP2;8 JePIP2;5 OsPIP2;6 AtPIP2;7, AtPIP2;8
    PIP2I CePIP2;9, CePIP2;10,
    CePIP2;11
    CcPIP2;10, CcPIP2;11 RbPIP2;9, RbPIP2;10 JePIP2;6 OsPIP2;7, OsPIP2;8
    At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effuses; Os: O. sativa; Rb: R. breviuscula; PIP: plasma membrane intrinsic protein.
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    Tissue-specific expression profiles of CePIP genes were investigated using transcriptome data available for young leaf, mature leaf, sheath, root, rhizome, shoot apex, and tuber. As shown in Fig. 3a, CePIP genes were mostly expressed in roots, followed by sheaths, moderately in tubers, young leaves, rhizomes, and mature leaves, and lowly in shoot apexes. In most tissues, CePIP1;1, -2;1, and -2;8 represent three dominant members that contributed more than 90% of total transcripts. By contrast, in rhizome, these three members occupied about 80% of total transcripts, which together with CePIP1;3 and -2;4 contributed up to 96%; in root, CePIP1;1, -1;3, -2;4, and -2;7 occupied about 84% of total transcripts, which together with CePIP2;1 and -2;8 contributed up to 94%. According to their expression patterns, CePIP genes could be divided into five main clusters: Cluster I includes CePIP1;1, -2;1, and -2;8 that were constitutively and highly expressed in all tissues examined; Cluster II includes CePIP2;2, -2;9, and -2;10 that were lowly expressed in all tested tissues; Cluster III includes CePIP1;2 and -2;11 that were preferentially expressed in young leaf and sheath; Cluster IV includes CePIP1;3 and -2;4 that were predominantly expressed in root and rhizome; and Cluster V includes remains that were typically expressed in root (Fig. 3a). Collectively, these results imply expression divergence of most duplicate pairs and three members (i.e. CePIP1;1, -2;1, and -2;8) have evolved to be constitutively co-expressed in most tissues.

    Figure 3.  Expression profiles of CePIP genes in various tissues, different stages of leaf development, and mature leaves of diurnal fluctuation. (a) Tissue-specific expression profiles of 14 CePIP genes. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) Expression profiles of CePIP1;1, -2;1, and -2;8 at different stages of leaf development. (c) Expression profiles of CePIP1;1, -2;1, and -2;8 in mature leaves of diurnal fluctuation. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p< 0.01). (Ce: C. esculentus; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein)

    As shown in Fig. 3a, compared with young leaves, transcriptome profiling showed that CePIP1;2, -2;3, -2;7, -2;8, and -2;11 were significantly down-regulated in mature leaves, whereas CePIP1;3 and -2;1 were up-regulated. To confirm the results, three dominant members, i.e., CePIP1;1, -2;1, and -2;8, were selected for qRT-PCR analysis, which includes three representative stages, i.e., young, mature, and senescing leaves. As shown in Fig. 3b, in contrast to CePIP2;1 that exhibited a bell-like expression pattern peaking in mature leaves, transcripts of both CePIP1;1 and -2;8 gradually decreased during leaf development. These results were largely consistent with transcriptome profiling, and the only difference is that CePIP1;1 was significantly down-regulated in mature leaves relative to young leaves. However, this may be due to different experiment conditions used, i.e., greenhouse vs natural conditions.

    Diurnal fluctuation expression patterns of CePIP1;1, -2;1, and -2;8 were also investigated in mature leaves and results are shown in Fig. 3c. Generally, transcripts of all three genes in the day (8, 12, 16, and 20 h) were higher than that in the night (24 and 4 h). During the day, both CePIP1;1 and -2;8 exhibited an unimodal expression pattern that peaked at 12 h, whereas CePIP2;1 possessed two peaks (8 and 16 h) and their difference was not significant. Nevertheless, transcripts of all three genes at 20 h (onset of night) were significantly lower than those at 8 h (onset of day) as well as 12 h. In the night, except for CePIP2;1, no significant difference was observed between the two stages for both CePIP1;1 and -2;8. Moreover, their transcripts were comparable to those at 20 h (Fig. 3c).

    To reveal the expression patterns of CePIP genes during tuber development, three representative stages, i.e., 40 DAS (early swelling stage), 85 DAS (late swelling stage), and 120 DAS (mature stage), were first profiled using transcriptome data. As shown in Fig. 4a, except for rare expression of CePIP1;2, -2;2, -2;9, and -2;10, most genes exhibited a bell-like expression pattern peaking at 85 DAS, in contrast to a gradual decrease of CePIP2;3 and -2;8. Notably, except for CePIP2;4, other genes were expressed considerably lower at 120 DAS than that at 40 DAS. For qRT-PCR confirmation of CePIP1;1, -2;1, and -2;8, seven stages were examined, i.e., 1, 5, 10, 15, 20, 25, and 35 DAI, which represent initiation, five stages of swelling, and maturation as described before[32]. As shown in Fig. 4b, two peaks were observed for all three genes, though their patterns were different. As for CePIP1;1, compared with the initiation stage (1 DAI), significant up-regulation was observed at the early swelling stage (5 DAI), followed by a gradual decrease except for the appearance of the second peak at 20 DAI, which is something different from transcriptome profiling. As for CePIP2;1, a sudden drop of transcripts first appeared at 5 DAI, then gradually increased until 20 DAI, which was followed by a gradual decrease at two late stages. The pattern of CePIP2;8 is similar to -1;1, two peaks appeared at 5 and 20 DAI and the second peak was significantly lower than the first. The difference is that the second peak of CePIP2;8 was significantly lower than the initiation stage. By contrast, the second peak (20 DAI) of CePIP2;1 was significantly higher than that of the first one (1 DAI). Nevertheless, the expression patterns of both CePIP2;1 and -2;8 are highly consistent with transcriptome profiling.

    Figure 4.  Transcript and protein abundances of CePIP genes during tuber development. (a) Transcriptome-based expression profiling of 14 CePIP genes during tuber development. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) qRT-PCR-based expression profiling of CePIP1;1, -2;1, and -2;8 in seven representative stages of tuber development. (c) Relative protein abundance of CePIP1;1, -2;1, and -2;8 in three representative stages of tuber development. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p < 0.01). (Ce: C. esculentus; DAI: days after tuber initiation; DAS: days after sowing; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein).

    Since protein abundance is not always in agreement with the transcript level, protein profiles of three dominant members (i.e. CePIP1;1, -2;1, and -2;8) during tuber development were further investigated. For this purpose, we first took advantage of available proteomic data to identify CePIP proteins, i.e., leaves, roots, and four stages of tubers (freshly harvested, dried, rehydrated for 48 h, and sprouted). As shown in Supplemental Fig. S2, all three proteins were identified in both leaves and roots, whereas CePIP1;1 and -2;8 were also identified in at least one of four tested stages of tubers. Notably, all three proteins were considerably more abundant in roots, implying their key roles in root water balance.

    To further uncover their profiles during tuber development, 4D-PRM-based protein quantification was conducted in three representative stages of tuber development, i.e., 1, 25, and 35 DAI. As expected, all three proteins were identified and quantified. In contrast to gradual decrease of CePIP2;8, both CePIP1;1 and -2;1 exhibited a bell-like pattern that peaked at 25 DAI, though no significant difference was observed between 1 and 25 DAI (Fig. 4c). The trends are largely in accordance with their transcription patterns, though the reverse trend was observed for CePIP2;1 at two early stages (Fig. 4b & Fig. 4c).

    As predicted by WoLF PSORT, CePIP1;1, -2;1, and -2;8 may function in the cell membrane. To confirm the result, subcellular localization vectors named pNC-Cam1304-CePIP1;1, pNC-Cam1304-CePIP2;1, and pNC-Cam1304-CePIP2;8 were further constructed. When transiently overexpressed in tobacco leaves, green fluorescence signals of all three constructs were confined to cell membranes, highly coinciding with red fluorescence signals of the plasma membrane marker HbPIP2;3-RFP (Fig. 5).

    Figure 5.  (a) Schematic diagram of overexpressing constructs, (b) subcellular localization analysis of CePIP1;1, -2;1, and -2;8 in N. benthamiana leaves. (35S: cauliflower mosaic virus 35S RNA promoter; Ce: C. esculentus; EGFP: enhanced green fluorescent protein; kb: kilobase; NOS: terminator of the nopaline synthase gene; RFP: red fluorescent protein; PIP: plasma membrane intrinsic protein).

    Water balance is particularly important for cell metabolism and enlargement, plant growth and development, and stress responses[2,19]. As the name suggests, AQPs raised considerable interest for their high permeability to water, and plasma membrane-localized PIPs were proven to play key roles in transmembrane water transport between cells[1,18]. The first PIP was discovered in human erythrocytes, which was named CHIP28 or AQP1, and its homolog in plants was first characterized in Arabidopsis, which is known as RD28, PIP2c, or AtPIP2;3[3,7,53]. Thus far, genome-wide identification of PIP genes have been reported in a high number of plant species, including two model plants Arabidopsis and rice[10,11,1317,5456]. By contrast, little information is available on Cyperaceae, the third largest family within the monocot clade that possesses more than 5,600 species[57].

    Given the crucial roles of water balance for tuber development and crop production, in this study, tigernut, a representative Cyperaceae plant producing high amounts of oil in underground tubers[28,30,32], was employed to study PIP genes. A number of 14 PIP genes representing two phylogenetic groups (i.e., PIP1 and PIP2) or 12 orthogroups (i.e., PIP1A, PIP1B, PIP1C, PIP2A, PIP2B, PIP2C, PIP2D, PIP2E, PIP2F, PIP2G, PIP2H, and PIP2I) were identified from the tigernut genome. Though the family amounts are comparative or less than 13–21 present in Arabidopsis, cassava (Manihot esculenta), rubber tree (Hevea brasiliensis), poplar (Populus trichocarpa), C. cristatella, R. breviuscula, banana (Musa acuminata), maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and switchgrass (Panicum virgatum), they are relatively more than four to 12 found in eelgrass (Zostera marina), Brachypodium distachyon, foxtail millet (Setaria italic), J. effuses, Aquilegia coerulea, papaya (Carica papaya), castor been (Ricinus communis), and physic nut (Jatropha curcas) (Supplemental Table S4). Among them, A. coerulea represents a basal eudicot that didn't experience the γ WGD shared by all core eudicots[50], whereas eelgrass is an early diverged aquatic monocot that didn't experience the τ WGD shared by all core monocots[56]. Interestingly, though both species possess two PIP1s and two PIP2s, they were shown to exhibit complex orthologous relationships of 1:1, 2:2, 1:0, and 0:1 (Supplemental Table S5). Whereas AcPIP1;1/AcPIP1;2/ZmPIP1;1/ZmPIP1;2 and ZmPIP2;1/AcPIP2;1 belong to PIP1A and PIP2A identified in this study, AcPIP2;2 and ZmPIP2;2 belong to PIP2H and PIP2I, respectively (Supplemental Table S5), implying that the last common ancestor of monocots and eudicots possesses only one PIP1 and two PIP2s followed by clade-specific expansion. A good example is the generation of AtPIP1;1 and -2;6 from AtPIP1;5 and -2;1 via the γ WGD, respectively[17].

    In tigernut, extensive expansion of the PIP subfamily was contributed by WGD (2), transposed (2), tandem (5), and dispersed duplications (3). It's worth noting that, two transposed repeats (i.e., CePIP1;1/-1;3 and CePIP2;1/-2;8) are shared by rice, implying their early origin that may be generated sometime after the split with the eudicot clade but before Cyperaceae-Poaceae divergence. By contrast, two WGD repeats (i.e., CePIP1;1/-1;2 and CePIP2;2/-2;4) are shared by C. cristatella, R. breviuscula, and J. effusus but not rice and Arabidopsis, implying that they may be derived from WGDs that occurred sometime after Cyperaceae-Poaceae split but before Cyperaceae-Juncaceae divergence. The possible WGD is the one that was described in C. littledalei[58], though the exact time still needs to be studied. Interestingly, compared with Arabidopsis (1) and rice (2), tandem/proximal duplications played a more important role in the expansion of PIP genes in tigernut (5) as well as other Cyperaceae species tested (5–6), which were shown to be Cyperaceae-specific or even species-specific. These tandem repeats may play a role in the adaptive evolution of Cyperaceae species as described in a high number of plant species[14,41]. According to comparative genomics analyses, tandem duplicates experienced stronger selective pressure than genes formed by other modes (WGD, transposed duplication, and dispersed duplication) and evolved toward biased functional roles involved in plant self-defense[41].

    As observed in most species such as Arabidopsis[10,1417], PIP genes in all Cyperaceae and Juncaceae species examined in this study, i.e., tigernut, C. cristatella, R. breviuscula, and J. effuses, feature three introns with conserved positions. By contrast, zero to three introns was not only found in rice but also in other Poaceae species such as maize, sorghum, foxtail millet, switchgrass, B. distachyon, and barley[54,55], implying lineage/species-specific evolution.

    Despite the extensive expansion of PIP genes (PIP2) in tigernut even after the split with R. breviuscula, CePIP1;1, -2;1, and -2;8 were shown to represent three dominant members in most tissues examined in this study, i.e., young leaf, mature leaf, sheath, rhizome, shoot apex, and tuber, though the situation in root is more complex. CePIP1;1 was characterized as a transposed repeat of CePIP1;3, which represents the most expressed member in root. Moreover, its recent WGD repeat CePIP1;2 was shown to be lowly expressed in most tested tissues, implying their divergence. The ortholog of CePIP1;1 in rice is OsPIP1;3 (RWC-3), which was shown to be preferentially expressed in roots, stems, and leaves, in contrast to constitutive expression of OsPIP1;1 (OsPIP1a) and -1;2[5961], two recent WGD repeats. Injecting the cRNA of OsPIP1;3 into Xenopus oocytes could increase the osmotic water permeability by 2–3 times[60], though the activity is considerably lower than PIP2s such as OsPIP2;2 and -2;2[6163]. Moreover, OsPIP1;3 was shown to play a role in drought avoidance in upland rice and its overexpression in lowland rice could increase root osmotic hydraulic conductivity, leaf water potential, and relative cumulative transpiration at the end of 10 h PEG treatment[64]. CePIP2;8 was characterized as a transposed repeat of CePIP2;1. Since their orthologs are present in both rice and Arabidopsis (Supplemental Table S3), the duplication event is more likely to occur sometime before monocot-eudicot split. Interestingly, their orthologs in rice, i.e., OsPIP2;1 (OsPIP2a) and -2;6, respectively, are also constitutively expressed[61], implying a conserved evolution with similar functions. When heterologously expressed in yeast, OsPIP2;1 was shown to exhibit high water transport activity[62,6466]. Moreover, root hydraulic conductivity was decreased by approximately four folds in OsPIP2;1 RNAi knock-down rice plants[64]. The water transport activity of OsPIP2;6 has not been tested, however, it was proven to be an H2O2 transporter that is involved in resistance to rice blast[61]. More work especially transgenic tests may improve our knowledge of the function of these key CePIP genes.

    Leaf is a photosynthetic organ that regulates water loss through transpiration. In tigernut, PIP transcripts in leaves were mainly contributed by CePIP1;1, -2;1, and -2;8, implying their key roles. During leaf development, in contrast to gradual decrease of CePIP1;1 and -2;8 transcripts in three stages (i.e. young, mature, and senescing) examined in this study, CePIP2;1 peaked in mature leaves. Their high abundance in young leaves is by cell elongation and enlargement at this stage, whereas upregulation of CePIP2;1 in mature leaves may inform its possible role in photosynthesis[67]. Thus far, a high number of CO2 permeable PIPs have been identified, e.g., AtPIP2;1, HvPIP2;1, HvPIP2;2, HvPIP2;3, HvPIP2;5, and SiPIP2;7[6870]. Moreover, in mature leaves, CePIP1;1, -2;1, and -2;8 were shown to exhibit an apparent diurnal fluctuation expression pattern that was expressed more in the day and usually peaked at noon, which reflects transpiration and the fact that PIP genes are usually induced by light[11,7173]. In rice, OsPIP2;4 and -2;5 also showed a clear diurnal fluctuation in roots that peaked at 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness[11]. Notably, further studies showed that temporal and dramatic induction of OsPIP2;5 around 2 h after light initiation was triggered by transpirational demand but not circadian rhythm[74].

    As an oil-bearing tuber crop, the main economic goal of tigernut cultivation is to harvest underground tubers, whose development is highly dependent on water available[32,75]. According to previous studies, the moisture content of immature tigernut tubers maintains more than 80.0%, followed by a seed-like dehydration process with a drop of water content to less than 50% during maturation[28,32]. Thereby, the water balance in developmental tubers must be tightly regulated. Like leaves, the majority of PIP transcripts in tubers were shown to be contributed by CePIP1;1, -2;1, and -2;8, which was further confirmed at the protein level. In accordance with the trend of water content during tuber development, mRNA, and protein abundances of CePIP1;1, -2;1, and -2;8 in initiation and swelling tubers were considerably higher than that at the mature stage. High abundances of CePIP1;1, -2;1, and -2;8 at the initiation stage reflects rapid cell division and elongation, whereas upregulation of CePIP1;1 and -2;1 at the swelling stage is in accordance with cell enlargement and active physiological metabolism such as rapid oil accumulation[28,30]. At the mature stage, downregulation of PIP transcripts and protein abundances resulted in a significant drop in the moisture content, which is accompanied by the significant accumulation of late embryogenesis-abundant proteins[23,32]. The situation is highly distinct from other tuber plants such as potato (Solanum tuberosum), which may contribute to the difference in desiccation resistance between two species[32,76]. It's worth noting that, in one study, CePIP2;1 was not detected in any of the four tested stages, i.e., freshly harvested, dried, rehydrated for 48 h, and sprouted tubers[23]. By contrast, it was quantified in all three stages of tuber development examined in this study, i.e., 1, 25, and 35 DAI (corresponding to freshly harvested tubers), which represent initiation, swelling, and maturation. One possible reason is that the protein abundance of CePIP2;1 in mature tubers is not high enough to be quantified by nanoLC-MS/MS, which is relatively less sensitive than 4D-PRM used in this study[30,46]. In fact, nanoLC-MS/MS-based proteomic analysis of 30 samples representing six tissues/stages only resulted in 2,257 distinct protein groups[23].

    Taken together, our results imply a key role of CePIP1;1, -2;1, and -2;8 in tuber water balance, however, the mechanism underlying needs to be further studied, e.g., posttranslational modifications, protein interaction patterns, and transcriptional regulators.

    To our knowledge, this is the first genome-wide characterization of PIP genes in tigernut, a representative Cyperaceae plant with oil-bearing tubers. Fourteen CePIP genes representing two phylogenetic groups or 12 orthogroups are relatively more than that present in two model plants rice and Arabidopsis, and gene expansion was mainly contributed by WGD and transposed/tandem duplications, some of which are lineage or even species-specific. Among these genes, CePIP1;1, -2;1, and -2;8 have evolved to be three dominant members that are constitutively expressed in most tissues, including leaf and tuber. Transcription of these three dominant members in leaves are subjected to development and diurnal regulation, whereas in tubers, their mRNA and protein abundances are positively correlated with the moisture content during tuber development. Moreover, their plasma membrane-localization was confirmed by subcellular localization analysis, implying that they may function in the cell membrane. These findings shall not only provide valuable information for further uncovering the mechanism of tuber water balance but also lay a solid foundation for genetic improvement by regulating these key PIP members in tigernut.

    The authors confirm contribution to the paper as follows: study conception and design, supervision: Zou Z; analysis and interpretation of results: Zou Z, Zheng Y, Xiao Y, Liu H, Huang J, Zhao Y; draft manuscript preparation: Zou Z, Zhao Y. All authors reviewed the results and approved the final version of the manuscript.

    All the relevant data is available within the published article.

    This work was supported by the Hainan Province Science and Technology Special Fund (ZDYF2024XDNY171 and ZDYF2024XDNY156), China; the National Natural Science Foundation of China (32460342, 31971688 and 31700580), China; the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-66), China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The authors declare that they have no conflict of interest.

  • Supplemental Fig. S1 Effect of LLA on the redox status of cancer cells. Mouse colon cancer C26 cells were treated by indicated concentrations of LLA for 24 h, after which the intracellular levels of GSH (A) and ROS (B) were quantified. Values are mean ± SD; n = 3. See text for details.
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    Gao S, Fang J, Zhou J, Yokomizo K. 2023. Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects. Food Materials Research 3:19 doi: 10.48130/FMR-2023-0019
    Gao S, Fang J, Zhou J, Yokomizo K. 2023. Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects. Food Materials Research 3:19 doi: 10.48130/FMR-2023-0019

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Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects

Food Materials Research  3 Article number: 19  (2023)  |  Cite this article

Abstract: In recent decades, natural products have gained increasing attention as potential therapies for chronic diseases, including inflammation and cancer. Among them, a hot water extract derived from seven traditional Chinese herbs (Lycii fructus, Crataegi fructus, Phyllanthi fructus, Chrysanthemi flos, Coicis semen, Ganoderma lucidum, and Zizyphi fructus), referred to as LLA, has demonstrated potent antioxidative effects. In line with this, our study aimed to investigate the anti-inflammatory properties of LLA and explore its potential for cancer prevention. To evaluate the anti-inflammatory effects of LLA, we employed a dextran sulfate sodium (DSS)-induced mouse colitis model. Daily oral administration of LLA significantly suppressed colitis symptoms, improving the disease activity index and mitigating colon tissue damage. Moreover, LLA treatment effectively inhibited the production of proinflammatory cytokines, including IL-6, TNF-α, and MCP-1. In addition to its anti-inflammatory effects, LLA exhibited remarkable efficacy in suppressing mouse colon carcinogenesis induced by azoxymethane (AOM) and DSS. Administration of LLA during the AOM/DSS treatment resulted in a significant reduction in tumor occurrence and growth, highlighting its potential as a preventive agent against colon cancer. Furthermore, we investigated the antitumor effect of LLA using a mouse sarcoma S180 solid tumor model. Although LLA did not demonstrate significant antitumor effects on established tumors, its application prior to tumor formation significantly inhibited tumor occurrence and growth. Collectively, these findings underscore the beneficial effects of LLA administration in the context of inflammatory diseases and highlight its potential as a preventive measure against cancer.

    • Cancer remains the leading cause of human death worldwide. The International Agency for Research on Cancer (IARC) 2020 report revealed approximately more than 19.3 million new cases of cancer patients, with breast cancer having the highest incidence (11.7%) followed by lung cancer (11.4%), colorectal cancer (10.0%), prostate cancer (7.3%), and gastric cancer (5.6%). Cancer is a multifactorial disease with many events involved in its initiation and progression, with inflammation being one of the major carcinogens. Notably, chronic inflammation can trigger DNA mutation and then carcinogenesis via reactive oxygen species (ROS) generation[1,2]. Thus, anti-oxidative, anti-inflammatory agents are considered useful for cancer prevention[2].

      Many natural products have various biological activities, including anti-inflammation, innate immunity activation, and anticancer effects[3,4]. Natural products have been used as medicines for many diseases in which inflammation and cancer are the major targets[5]. For example, many conventional anticancer drugs, such as paclitaxel, camptothecin, and vincristine, originate from natural products[3,4]. The development and utilization of natural products for controlling inflammatory diseases and cancer has become a research hotspot. In this context, we have discovered canolol, a potent antioxidative, anti-inflammatory active component of rapeseed, which showed strong activity for removing ROS[6] and a remarkable suppressive effect against mouse ulcerative colitis induced by dextran sulfate sodium (DSS), and thus significantly inhibited colorectal cancer carcinogenesis triggered by azoxymethane (AOM)/DSS[7]. We have also investigated the activity of hot water extract of Phellinus linteus, fresh leaves of Kumaizasa bamboo, and Chaga mushroom (i.e., MeshimaMax) and found a remarkable cancer-preventive effect in both transplanted tumor and carcinogen (AOM/DSS, 7,12-dimethylbenz[a]anthracene)-induced colon cancer, which is strongly associated with the activation of macrophage and innate immunity[8]. These studies suggest potential applications of natural products for cancer prevention.

      Along this line, the current study focuses on a hot water extract of seven traditional Chinese medicines (Lycii fructus, Crataegi fructus, Phyllanthi fructus, Chrysanhemi flos, Coicts semen, Ganoderma lucidum, and Zizyphi fructus), which is referred to as LLA. As a nutritional supplement drink, LLA has been used in China and Japan for more than 30 years, and its main active components are flavonoids, organic acids, and polysaccharides. Notably, phyllanthi has been shown to exhibit anti-inflammatory and antipyretic effects, and its air-dried fruit has also been used to treat cancer in Tibetan and Egyptian medicines[9]. An in vitro study using mouse melanoma B16F10 cells showed that the extracts from the roots of phyllanti strongly inhibited cell growth and differentiation[5]. Moreover, Kanglaite injection, which originates from the oil extract of Coicts semen, is widely used for treating many cancers, including pancreatic, lung, gastric, and breast[1013]. In addition, improved superoxide dismutase (SOD) activity has been reported for polysaccharides derived from Lycium Chinese, a component of LLA, with vitamin C-like antioxidant activity[14,15].

      Though many studies suggested the anti-inflammatory and anti-cancer potential of the active components in LLA as described above, very few studies have been carried to explore the beneficial effects of the whole LLA as a nutritional drink. In a previous study using LLA, we found a remarkable ergogenic capacity in aged mice, and the effect was considered mostly due to the increased anti-oxidative activity, i.e., increased superoxide dismutase (SOD) activity[16]. It is well-recognized that the anti-oxidative activity is positively related to anti-inflammation and cancer-prevention potential[1,2,17,18]. Our previous studies using the extracts of natural products[68], as well as ample studies from other research groups[1921] strongly indicated that natural products with high anti-oxidative activity exert the beneficial potential for suppressing inflammation and cancer. Along this line, we considered that LLA may also exhibit anti-inflammation and cancer prevention potential, and this hypothesis was verified by using various solid tumor models, including chemical carcinogen-induced cancer that mimics the process of human cancer development. We also examined the possible mechanisms of action involved in the activity of LLA, focusing on its anti-inflammatory properties.

    • DSS was obtained from MP Biomedicals, LLC (Irvine, CA, USA). Fujifilm Wako Pure Chemical Corporation (Osaka, Japan) supplied AOM, cell culture medium (RPMI-1640, DMEM), Quantikine ELISA kit of mouse monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). LLA was obtained from the Institute of International Kampo Co. Ltd. While Sigma-Aldrich Chemical (St. Louis, MO, USA) supplied thiazolyl blue tetrazolium bromide (MTT) and 2′,7′-dichlorofluorescein diacetate (DCDHF-DA), Dojindo Molecular Technologies, Inc. (Kumamoto, Japan) supplied GSSG/GSH quantification kit.

    • Six-week-old male ddY mice and five-week-old male ICR mice were obtained from SLC (Shizuoka, Japan). The mice were housed at 22 ± 10 °C and 55% ± 5% relative humidity with automatic lighting at a 12-h light/dark cycle. Mice were fed adaptively for 1 week before experiments. During experiments, the mice were randomly divided into different groups as indicated.

    • A mouse sarcoma S180 tumor model was prepared by injecting S180 tumor cells (2 × 106 cells/100 uL) into the dorsal skin of a ddY mouse. LLA (0.07%, 0.2%, 0.6%) was given through drinking water; in one group, LLA was administered daily from the day of S180 cell inoculation, and in another group, daily LLA administration was carried out one week after tumor inoculation when the tumor grew to 6–8 mm in diameter. Each group included four mice. During the experiment, the width (W) and length (L) of the tumors, as well as the body weight of mice, were measured every 2–3 d during the study period, and tumor volume (mm3) was calculated as (W2 × L)/2. When the tumor grew to 4,000–5,000 mm3, the mice were euthanized by inhaling isoflurane at saturated vapor pressure.

    • A carcinogen-induced mouse colon cancer model was prepared by injecting AOM (10 mg/kg) i.p. into ICR mice, followed by daily administration of 2% DSS through drinking water from one week after AOM injection for 7 d. Each group included 6−8 mice. During the experiments, LLA in commonly used doses as a healthy supplement (i.e., 0.07%, 0.2%, 0.6%) was given through drinking water; however, during DSS treatment, LLA was given via food to avoid potential interference. At 12–13 weeks after AOM administration, the mice were sacrificed by isoflurane euthanasia as described above, colon and blood were collected, and the number and size of colon cancer nodules were measured.

    • The mouse colitis model was established by feeding 2% DSS to ICR mice in drinking water. LLA (0.07%, 0.2%, 0.6%) treatment was carried out throughout the experiment by adding LLA to the mouse food. Each group included 7−8 mice. During the experiments, we observed the symptoms and severity of colitis, which were evaluated using a semi-quantitative disease activity index (DAI). The DAI was determined by score changes in the body weight of mice, the presence of occult blood, gross bleeding, and stool consistency[22]. The weight loss of mice was graded into five levels (0: either a weight gain or no weight loss; 1: 1%–5% loss; 2: 5%–10% loss; 3: 10%–20% loss; and 4: more than 20% loss). Three levels of grade were used for stool consistency (0: normal; 2: loose; and 4: diarrhea) and occult blood (0: negative; 2: occult blood-positive; and 4: gross bleeding).

      On the 12th day after feeding DSS, when severe symptoms were observed, the mice were sacrificed by isoflurane euthanasia as described above, and the blood and colon specimens were collected for biochemical and pathological examinations. Serum samples were used to measure the inflammatory cytokines: MCP-1, TNF-α, and IL-6.

    • The cytotoxicity of LLA was examined in both cancer cells (human cervical cancer cells A2780) and normal cells (monkey kidney epithelial cells, CCL-81). The cells were cultured in RPMI-1640 containing 10% fetal bovine serum. The cells were seeded in 96-well plates (3,000 cells/well); after overnight incubation, different doses of LLA were added, and the cells were further cultured for 48 h. Cell viability was measured using an MTT assay[23].

    • The effect of LLA on the phagocytotic activity of macrophages was investigated using mouse macrophage RAW264.7 cells. RAW264.7 cells (1 × 105 cells/1.5 mL) were cultured on a 6-well plate; after overnight incubation, LLA of different concentrations were added and the cells were further cultured for 24 h. Then, yeast cells (Saccharomyces cerevisiae) (3 × 105 cells) were added to the culture media. At the scheduled time, the macrophages incorporating yeast were detected and counted using a microscope (BZ-X700, Keyence Co. Ltd., Osaka, Japan). Two hundred RAW264.7 cells were counted, and the phagocytic ability of macrophages was calculated by the following equation, phagocytosis = A / 200 × 100%, in which A was the number of macrophages that took up yeast cells.

    • Colon cancers were seeded in a 12-well plate (3.5 × 105 cells/well). After overnight pre-incubation, the indicated concentrations of LLA were added and the cells were treated for 24 h. The cells were then trypsinized and collected, and the glutathione (GSH) concentrations in the cells were quantified using a GSSG/GSH quantification kit (Dojindo Molecular Technologies, Inc.) following the manufacturer's instructions.

      For intracellular ROS measurement, C26 cancer cells were seeded in 12-well plates at a density of 2 × 105 cells/well and incubated for 12 h. Different concentrations of LLA were then added to the cells, and the cells were treated for 24 h. To detect intracellular ROS, a 10 μM solution of the fluorescent probe DCDHF-DA was added to the cells 30 min before harvesting. Upon internalization, DCDHF-diacetate is converted to DCDHF, which reacts with ROS to form the fluorescent compound dichlorofluorescein. Subsequently, the cells were exposed to 100 μM hydrogen peroxide (H2O2) for 30 min. The level of intracellular ROS was quantified by measuring the fluorescence intensity using flow cytometry (BD AccuriTM C6 Plus; Becton Dickinson, San Jose, CA, USA). In some experiments, LLA was administered to the cells simultaneously with H2O2 exposure. In a separate study, the same protocol was carried out without H2O2 exposure.

    • All data were expressed as mean ± SD. Data were analyzed using ANOVA, followed by the Bonferroni multiple comparison test. A difference was considered statistically significant when p < 0.05.

    • We first investigated the anti-inflammatory activity of LLA using a DSS-induced mouse inflammatory colitis model. As shown in Fig. 1a, after feeding DSS for 5 d, colitis was triggered and rapidly progressed, as evidenced by the increased DAI. Histological examination confirmed colitis formation, as evidenced by colon shortening (Fig. 1b), and destruction of colon mucosa (Fig. 1c). In parallel with these findings, a remarkable increase in inflammatory cytokines (i.e., TNF-α, MCP-1, and IL-6) was observed (Fig. 2). LLA administration significantly suppressed the above-described symptoms and histopathological changes in colitis (Fig. 1). Notably, after LLA treatment, inflammatory cytokine generation was largely inhibited, and the levels of all tested inflammatory cytokines decreased to almost normal levels at all tested doses (Fig. 2).

      Figure 1. 

      Anti-inflammatory effect of LLA on DSS-induced mouse colitis. The DSS-induced colitis model was triggered by oral administration of 2% DSS. LLA treatment was carried out throughout the experiment. During the experiments, symptoms of colitis were recorded daily, and (a) the disease activity index (DAI) values were calculated. On day 11, when severe colitis appeared, mice were killed, (b) the length of the colon was measured, and (c) histological examination of the colon was carried out. Values are mean ± SD; n = 7–8. *, p < 0.05; **, p < 0.01 vs DSS colitis group. See text for details.

      Figure 2. 

      (a) Inhibition of inflammatory cytokines TNF-α, (b) MCP-1, and (c) IL-6 by LLA in DSS-induced murine colitis. The experimental protocol is the same as that described in Fig. 3. On day 11 of the experiment, mice were killed, and serum samples were collected for measurement of the cytokines by using ELISA. Values are mean ± SD; n = 7–8. *, p < 0.05; **, p < 0.01; *, ***, p < 0.001. See text for details.

    • In the DSS induced colitis model, as expected we clearly found the suppression of inflammation by LLA. It is thus reasonable to anticipate the potential effects of LLA for cancer, we thus performed the studies using two solid tumor models as below.

    • In the mouse sarcoma S180 tumor model, we first examined the cancer-preventive effect of LLA by starting the LLA treatment on the same day that the S180 cells were injected into the mice. As shown in Fig. 3a, tumor growth was significantly inhibited dose-dependently.

      Figure 3. 

      Antitumor effect of Ligustrum lucidum ait (LLA) against mouse sarcoma S180 solid tumor. (a) LLA was fed to the mice from the day of mouse sarcoma S180 tumor cell inoculation. (b) LLA was fed to the mice from day 7 after S180 tumor cell inoculation when the tumor had grown to 7–8 mm in diameter. Arrows indicate the date to start the LLA treatment. Data are mean ± SD, n = 4. *, p < 0.05, 0.6% LLA group vs Control. See text for details.

      However, when LLA treatment was initiated one week after tumor inoculation when the tumor grew to 7–8 mm in diameter, we did not find significant inhibition of tumor growth, although 0.2% of LLA showed an apparent trend of tumor growth inhibition (Fig. 3b). These findings suggest that LLA does not exhibit direct tumoricidal activity; that is, LLA as a healthy supplement drink did not show a significant anticancer effect.

    • We then focused on the cancer-preventive effect of LLA and confirmed it using a carcinogen (AOM/DSS)-induced mouse colon cancer model. In line with the results shown in Fig. 3a, we found remarkably decreased tumor nodules in the colon (Fig. 4). However, the LLA effect was not dose-dependent: 0.2% of LLA exhibited a better effect than 0.6% (Fig. 4).

      Figure 4. 

      Suppression of azoxymethane/dextran sulfate sodium (AOM/DSS) induced mouse colon carcinogenesis by LLA. At 12-13 weeks after AOM administration with/without LLA treatment, mice were killed and the numbers of tumor nodules in the colon were counted (left), and the photos of the colon were shown on the right Data are mean ± SD, n = 9. *, p < 0.05 vs control. See text for details.

    • To further tackle the actions and mechanisms of LLA against cancer, we then measured the in vitro cytotoxicity of LLA using both tumor and normal cells. In both normal (CCL-81) and tumor cells (C26 cells), almost no or very little cytotoxicity of LLA was found, up to 1 mg/ml (Fig. 5), however, remarkable cytotoxicity was observed at concentration higher than 1 mg/ml). These findings, along with its anti-inflammatory, cancer preventive effect as shown in Figs 1 to 4, indicate the inverted U-shape dose effect of LLA.

      Figure 5. 

      (a) In vitro cytotoxicity of LLA to normal cells (monkey kidney epithelial cells CCL-81, as well as (b) tumor cells (human ovarian cancer A2780). Cell viability after LLA treatment was examined by MTT assay. Values are mean ± SD; n = 8. See text for details.

    • Our previous study showed that extracts of natural products exhibited an antitumor effect by activating innate immunity[8]. Therefore, we measured the effect of LLA on the phagocytotic activity of macrophages. As shown in Fig. 6, macrophages gradually absorbed yeast added to the culture medium time-dependently; however, LLA treatment did not increase the phagocytosis rate of macrophages, suggesting that macrophage activation does not serve as a mechanism in LLA-induced cancer prevention.

      Figure 6. 

      Effect of LLA on the phagocytotic activity of macrophage. Engulfment of yeast by macrophages in the presence /absence (control) of LLA was observed and counted with a microscope for 12 h after adding yeast to macrophages, the phagocytosis rate of macrophages was then calculated. Values are mean ± SD; n = 3. See text for details.

    • A previous study using LLA suggested the antioxidative potential of LLA, that is, increased SOD activity and GSH levels in the mice[16]. To determine whether LLA could affect the redox status of cancer cells, we measured the antioxidant GSH and ROS levels in colon cancer C26 cells after LLA treatment. No apparent changes in GSHand ROS (Supplemental Fig. S1) were observed after LLA treatment up to 1 mg/ml. However, upon exposure of cells to hydrogen peroxide (H2O2) to induce ROS generation, we observed that LLA treatment effectively suppressed intracellular ROS levels in a dose-dependent manner (Fig. 7). This effect was observed both when LLA was administered as a pre-treatment (Fig. 7a) and when it was co-exposed with H2O2 (Fig. 7b). These results strongly suggest that LLA does not disrupt the redox homeostasis of the cells but instead exerts robust anti-oxidative effects in the face of oxidative stress.

      Figure 7. 

      Suppression of intracellular ROS induced by hydrogen peroxide (H2O2) through LLA treatment in C26 colon cancer cells. (a) Pre-treatment: C26 cells were treated with increasing concentrations of LLA for 24 h prior to H2O2 addition. (b) Co-exposure: LLA was administered simultaneously with H2O2 exposure. The intracellular ROS levels were quantified using the fluorescence ROS probe DCDHF-DA and analyzed by flow cytometry. Mean values ± standard deviation (SD) are shown; n = 4–8. *, p < 0.05, **, p < 0.01 compared to the H2O2 alone group. See text for details.

    • LLA is a well-recognized supplemental drink that has been used in Japan and China for decades. We previously found that LLA significantly increased SOD and GSH levels in the spleen and liver[16], suggesting the antioxidative activity of LLA. Notably, ROS plays an important role in the progression of inflammation, which leads to the cell's malignant transformation. Moreover, polyphenols, which are the major effector molecules in fruits, exhibit cancer-preventive effects, mostly through their anti-inflammatory properties[24]. As a result, it is reasonable to speculate that LLA may have an anti-inflammatory effect and thus aid in cancer prevention. As expected, we found that LLA exhibited a potent anti-inflammatory effect in the DSS-induced mouse inflammatory colitis model (Fig. 1), in which the symptoms and pathological changes were remarkably improved by LLA (Fig. 1), and inflammatory cytokine production was significantly suppressed (Fig. 2). In line with the anti-inflammatory activity of LLA, carcinogenesis induced by AOM/DSS, which is a typical model of inflammation-related carcinogenesis, was markedly inhibited by LLA administration (Fig. 4), indicating the strong potential of LLA for cancer prevention.

      In this study, we did not find a direct anticancer effect of LLA: feeding LLA to mice bearing solid tumors did not suppress tumor growth significantly (Fig. 3b). However, previous studies have indicated the antitumor activities of traditional Chinese medicine in LLA. For example, the fruit extract of Phyllanthus emblica has long been used in traditional medicine to treat many diseases, such as constipation and cancer[25]. From the leaves, roots, and fruit juice of Phyllanthus emblica, 18 main components have been extracted and identified, such as phyllaemblicin B, phyllaemblicin C, and phyllemtannin, most of which showed potent tumoricidal effects against many tumor cells[5]. In addition, the oil extract of Coicts semen, Kanglaite injection, not only shows an anticancer effect but can also largely enhance the host's immune function, which was approved in 1997 by the Ministry of Health of China for treating many cancers: over millions of cancer patients in more than 2,000 hospitals in China have been treated with Kanglaite injection[10,26]. It is not clear why LLA had no significant anticancer effect, but we believe that dosing is the main reason. Namely, the above-mentioned studies used a condensed extract of traditional medicines at relatively high concentrations; however, the LLA used in our present study was a supplement drink containing the hot water extract of seven Chinese traditional medicines, which was used and evaluated as a whole as a healthy supplement but not as a pharmaceutical agent. This means that the active components in LLA that may have exhibited tumoricidal effects were very limited. In addition, the dosing in this study was comparable to the commonly suggested amount of this supplement drink (e.g., the dose of 0.6% is comparable to 135 ml per day in humans). Thus, by the protocol used in this study, the effector components with anticancer effects could not reach or were much lower than the cytotoxic doses. Future studies are warranted to identify the anticancer components of LLA and to elucidate the potential of LLA for cancer treatment.

      Although we did not find an apparent anticancer effect of LLA, its potential for cancer prevention was found. LLA administration before tumor formation significantly inhibited tumor occurrence and growth (Fig. 3a), and feeding LLA to the mice remarkably suppressed the colon cancer carcinogenesis triggered by carcinogen AOM/DSS (Fig. 4) that mimics the process of human cancer development. Regarding the possible mechanisms of action for the cancer-preventive effect of LLA, we did not find an apparent cytotoxic activity of LLA (Fig. 5) or observe its effect on macrophage activation (Fig. 6), suggesting that cytotoxicity and activation of innate immunity are not involved in the cancer-preventive effect of LLA. In a previous study using LLA, our group found that LLA exhibited a potent antioxidative effect by increasing the activity of the antioxidative enzyme SOD and the amount of antioxidant GSH[16], indicating the anti-inflammatory potential of LLA. As expected, we found a remarkable anti-inflammatory effect of LLA in the DSS-induced mouse colitis model (Figs 3, 4). Unexpectedly, we did not observe significant changes in the cellular redox status, as measured by the levels of GSH and ROS, in cancer cells after LLA treatment under normal conditions (Supplemental Fig. S1). However, we made an important discovery when examining the response of cells to oxidative stress. LLA treatment significantly suppressed the generation of intracellular ROS when the cells were exposed to H2O2 (Fig. 7), indicating the potent anti-oxidative effect of LLA under conditions of pathological oxidative stress. Given that inflammation plays a major role in cancer development, and ROS is a critical factor in triggering inflammation and promoting cancer initiation and progression, we propose that LLA's cancer-preventive effect is largely attributed to its anti-oxidative and anti-inflammatory activities. Nonetheless, further investigations are warranted to elucidate the precise mechanisms underlying the action of LLA, and we plan to conduct these investigations in future studies.

      Furthermore, while we observed LLA's anti-inflammatory and cancer-preventive effects, we discovered that the effect was not linearly dose-dependent. In both studies using the DSS-induced colitis model and AOM/DSS-induced mouse colon carcinogenesis model, 0.2% LLA showed the best effect, overwhelming both 0.07% and 0.6% (Figs 13). These findings provide evidence for an inverted 'U-shaped' dose response effect of LLA, where low to moderate concentrations exhibit anti-inflammatory properties, while higher doses may induce proinflammatory and cytotoxic effects. This observation may be attributed to the tumoricidal effects of the individual components within LLA, as discussed earlier. Interestingly, our in vitro cytotoxicity study revealed a striking discovery: LLA demonstrated a remarkable cytotoxic effect at concentrations exceeding 1 mg/ml (Fig. 5). This finding sheds light on the inverted U-shaped dose-response relationship of LLA and its potential implications. Furthermore, we found a similar phenomenon in our previous study using an extract mixture of Phellinus linteus, bamboo leaf, and chaga mushroom[8], and other researchers also reported similar findings using natural herbs[27]. However, the detailed mechanisms underlying this dose effect remain unclear, necessitating further investigations.

      Although the cytotoxicity of LLA components has been reported in many cancer cells[5], we found no evidence of LLA cytotoxicity up to 1 mg/kg in either cancer or normal cells (Fig. 5). While these findings support our previously stated hypothesis that LLA's cancer-preventive effect is not due to its tumoricidal effect, they also support LLA's safety/nontoxicity as a supplemental drink.

      Nowadays, various healthy supplements, including supplemental drinks originating from natural products and traditional Chinese medicines, have been used habitually for health-oriented purposes. While most studies regarding healthy supplements focus on the effect of a single component and active molecule in a supplement, it is also crucial to evaluate the compositive effect of the supplement as a whole under routine doses and usage. We thus believe that the current study provides insights into the effect of LLA and its future applications.

    • We have revealed that the extract of seven traditional Chinese medicines, LLA, had potent cancer-preventive activity not only in the transplanted solid tumor model but also in the carcinogen-induced colon carcinogenesis model, which mimics the actual initiation and progression of human cancer. The cancer-preventive effect of LLA is mostly the consequence of suppressing inflammation. We believe that this is the first study regarding the potential of LLA for cancer prevention, and we anticipate that this supplement drink will benefit patients suffering from inflammatory diseases as well as be useful for cancer prevention.

    • All experiments were conducted following the Laboratory Protocol of Animal Handling, Sojo University, and were approved by the Animal Ethical Committee, Sojo University (No. P/009/2019).

      • This research was partly funded by the research funding from Faculty of Pharmaceutical Sciences, Sojo University to Jun Fang and Kazumi Yokomizo.

      • The authors declare that they have no conflict of interest.

      • These authors contributed equally: Shanghui Gao

      • Supplemental Fig. S1 Effect of LLA on the redox status of cancer cells. Mouse colon cancer C26 cells were treated by indicated concentrations of LLA for 24 h, after which the intracellular levels of GSH (A) and ROS (B) were quantified. Values are mean ± SD; n = 3. See text for details.
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press on behalf of Nanjing Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
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  • About this article
    Cite this article
    Gao S, Fang J, Zhou J, Yokomizo K. 2023. Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects. Food Materials Research 3:19 doi: 10.48130/FMR-2023-0019
    Gao S, Fang J, Zhou J, Yokomizo K. 2023. Extract essence of seven Chinese herbs potentially exhibit anti-inflammatory and cancer-preventive effects. Food Materials Research 3:19 doi: 10.48130/FMR-2023-0019

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