Population genetic studies provide a foundation for conservation planning, especially for endangered species. Three chloroplast SSRs (mtrnSf-trnGr, mtrnL2-trnF, and mtrnL5-trnL3) and the internal transcribed spacer were used to examine the population structure of Helianthemum in northwestern China. A total of 15 populations of the genus were collected. Nine chloroplast haplotypes and two nuclear genotypes were detected. Both the nuclear and chloroplast data showed two lineages in Helianthemum songaricum, respectively, distributed in Yili Valley and western Ordos Plateau. A total of 66.81% (p < 0.001) of the genetic variation was supported by this lineage split. A Mantel test showed a significant correlation between genetic distance and geographical distance (r = 0.937, p < 0.001). Based on genetic analyses, cpSSRs data support strong genetic divergence between regions. We speculate that the climate change during the late Tertiary and early Quaternary isolated H. songaricum into their current distribution, resulting in interruption of gene flow, leading to isolation and genetic divergence between the two regions. Meanwhile, possible selfing would increase genetic drift in small fragmented populations, that might account for the observed genetic divergence in both regions. Given the loss of genetic diversity and genetic divergence in small populations of Helianthemum in northwestern China immediate conservation management steps should be taken on the species.
Keywords: Helianthemum, Yili Valley, western Ordos Plateau, genetic diversity, genetic structure, conservation implication
Helianthemum is a shrub or subshrub mostly distributed in the Mediterranean, extending to Central Asia (Yang and Michael, 2007). Helianthemum songaricum and H. ordosicum in northwestern China is disjunctively distributed in Yili Valley of Xinjiang and western Ordos Plateau of Inner Mongolia, growing in rocky hills and slopes in steppe-desert regions between 1000 and 1400 m. It has spine-tipped branches, stipulate leaves, yellow flowers, and insect pollinated. Because of anthropogenic activities, such as grazing, mining, and the heavy harvest of firewood over the last few decades, H. songaricum and H. ordosicum has been in decline and become highly fragmented. As a result, it was listed as endangered in the China Species Red List (Fu, 1992).
There is disagreement with the taxonomic status of species in Helianthemum in northwestern China. Initially, taxonomists considered only one species of Helianthemum in northwestern China, Helianthemum songaricum Schrenk (Li, 1990). However, later studies found that there was a significant difference of pollen morphology and chromosome number between populations in Yili Valley and those in western Ordos Plateau. In the Yili Valley pollen was striate with a cytotype of 2n = 20, and in the western Ordos Plateau the pollen was perforate with 2n = 40 (Mo et al., 1997; Cao et al., 2000). Based on these results, a new taxon, H. ordosicum, was proposed in western Ordos (Zhao et al., 2000). More recently, Yang and Michael (2007) recognized only one species, H. songaricum. In our previous phylogeographic study, two chloroplast intergenic spacers data supported two species: H. songaricum and H. ordosicum. (Su et al., 2011).
Correctly defining the taxonomy and populations (i.e., intraspecific manage units, MU) is essential to make effective conservation strategies for endangered species (Frankham et al., 2002). Incorrect taxonomy would lead to ill-conceived management strategies. For example, outcrossing of different taxa can create inviable or infertile offspring (Barton and Hewitt, 1981; Coyne and Orr, 1989) and dismantle of coadapted complexes (Mayr, 1963; Shields, 1982; Templeton, 1987), leading to outbreeding depression. Outcrossing depression can also be observed in crosses at the intraspecific level (Geiger, 1988; Waser and Price, 1989). Populations that have adapted to different habitats could also suffer from outbreeding depression and should treated as a distinct manage unit to avoid outcrossing (Frankham et al., 2002).
Information on genetic structure can resolve ambiguous classifications and establish a foundation for conservation genetic planning. Chloroplast simple sequence repeats (cpSSRs) are a highly polymorphic molecular tool in the population genetic analysis (Vendramin et al., 1996; Morgante et al., 1997; Ebert and Peakall, 2009). Besides their high mutation rates, they have other specific features. Because of their uniparental inheritance, they could show pronounced level of population differentiation (Ennos, 1994; Vendramin et al., 1999; Flannery et al., 2006). In addition, in monoecious species, uniparentally inherited genomes have only half the effective population size (Birky, 1988), therefore, these genomes are sensitive to historical bottlenecks (Morgante et al., 1997). Though the apparent advantages, chloroplast DNA markers might only provide partly genetic information of a species (Mäder et al., 2010), and combination with biparentally inherited nuclear DNA markers would present a more integral view of population structure and demography history (Burban and Petit, 2003; Petit et al., 2005).
The primary conclusion in our previous study, that was significant genetic divergence existed between Yili Valley and western Ordos Plateau, was only based on two chloroplast spacers (trnD-trnT and rps16-trnK) and need to be further improved with nuclear genome data. In addition, population structure in the two regions are still unclear because of the limited polymorphism in the two chloroplast spacers (Su et al., 2011). Here, we use three highly polymorphic cpSSRs and nuclear Internal Transcribed Spacer (ITS) sequence to investigate the full genetic structure of Helianthemum in northwestern China to address the following questions: (1) Whether populations of Helianthemum in western Ordos Plateau represent a distinct taxa, H. ordosicum? (2) if so, what is the genetic structure within the two species? (3) What are the conservation implications from the genetic structure analysis?
Materials and methods
In 2010 and 2014, H. songaricum and H. ordosicum was sampled throughout its distribution in northwestern China. A total of 15 populations of the species were collected: nine from Yili Valley and six from western Ordos Plateau (Figure 1A). The geographical locations of the collection sites are presented in Table 1. Six to twelve individuals were sampled in each population. Fresh leaves were dried in silica gel and stored at 4°C until DNA extraction.
Sampling distribution of Helianthemum in China (a), the cp haplotype distribution (b), and ITS genotypes distribution in Helianthemum(c). Population numbers correspond to those in Table 1; cp haplotypes to those in Table ...
Details of sample locations, sample size, and genetic variation for 15 populations of Helianthemum.
DNA extraction, CpSSRS, and ITS sequencing
Total genomic DNA was extracted from dried leaves by the CTAB method (Doyle and Doyle, 1987). Polymerase chain reactions (PCR) were carried out in a volume of 25 μL reaction mixtures containing 4 mM MgCl2, 0.2 mM dNTP, 0.5 μmol primer, and 1 U Taq polymerase (Applied Biosystems, Foster City, Calif.), implemented in a Biorad T100 thermocycler (Biorad). The cpSSRs were amplified using three Helianthemum-specific polymorphic loci detected in regions trnL-trnF, trnL5-trnL3, and trnS-trnG: mtrnSf-trnGr, mtrnL2-trnF, and mtrnL5-trnL3 (see Soubani et al., 2014), and following the temperature profile: 95°C for 4 min; 30 cycles of 92°C for 45 s; 57°C for 45 s; and 72°C for 1 min; linked a extension at 72°C for 10 min; ITS2 region was amplified using primers of Sun et al. (1994), and following the temperature profile: 94°C for 5 min; 35 cycles of 94°C for 30 s; 52°C for 45 s; and 72°C for 1 min; linked a extension at 72°C for 8 min. The cpSSRs products were separated by capillary electrophoresis, with an ABI 3730xl (Applied Biosystems) automated sequencer. CpSSRs fragment sizes were determined in Geneious version 7.0 using the package Plugin (Kearse et al., 2012), using Gene-flo 625 (Chimerx) as the internal lane standard. ITS2 amplified primers were used in sequencing reactions conducting in the DYEnamic ET Terminator Kit (Amersham Pharmacia Biotech). Sequencing were carried out in ABI 3730xl. ITS2 electropherograms were edited and assembled in SEQUENCHER 4.8 (Gene Codes, Ann Arbor, MI, USA), then the sequences were aligned in CLUSTALW (Thompson et al., 1994), and refined by visual inspection.
Population genetic analysis
For ease of presentation in this study, the terms “locus” refers to a cpSSR site, and “allele” refers to a length-variant at a cpSSR site. Alleles of the three plastid loci were scored, respectively, treated as ordered characters and then combined together as multilocus haplotypes, assuming a stepwise pattern in mutation (Ohta and Kimura, 1973). Using stirling probability distribution and Bayes's theorem, the completeness of haplotype sampling in this study was estimated (Dixon, 2006).
The number of different alleles (Na), the effective number of alleles (Ne), and Nei's genetic distence (Nei, 1978), were caculated in GenAlEx 6.5 software (Peakall and Smouse, 2006). Within-population diversity (hS), total gene diversity (hT), genetic differentiation index (GST, leaves out mutation steps between haplotypes; NST, includes mutation steps between haplotypes) were calculated in the program HAPLONST, using U-test to determine whether NST is significantly larger than GST.
Using pairwise population differentiation measures (FST) as the variance components (Wright, 1965), analysis of molecular variance (AMOVA) was performed to study the partition of total genetic variation within and among populations, conducted in ARLEQUIN v.3.01 (Excoffier et al., 1992). The significance test used 10,000 permutations. To evaluate the population genetic structure, a Neighbor-Joining network (NJ) of the 15 populations was constructed in MEGA 6.0 (Tamura et al., 2013), using Nei's genetic distance matrix. This genetic distance matrix was also used to perform principal coordinate (PCO) analysis in GenAlEx 6.5 (Peakall and Smouse, 2006). To reveal the genetic divergence between the two regions, a Mantel test was performed in ARLEQUIN v.3.01, with 10,000 permutations significance test. Geographical distance was calculated in GEODIS 2.5 (Posada et al., 2000), natural-log transformed in Excel 2000, and then correlated with the Nei's genetic distances.
To analyse the genealogical relationships among all the chloroplast haplotypes, a network was constructed using median-joining method conducted in NETWORK v. 4.600 (Bandelt et al., 1999).
Allele and sequence analysis
A total of 13 alleles were detected in the three cpSSRs: three alleles in mtrnSf-trnGr, four alleles in mtrnL2-trnF, and six alleles in mtrnL5-trnL3. The 154 individuals sampled from 15 populations yielded 9 haplotypes (Table 2). Using the method described in Dixon (2006), the estimated probability of haplotype completeness was 1.0, suggesting that we have sampled almost all potential haplotypes in this study. For the ITS2 region, the aligned sequence length was 449 bp, and one informative nucleotide substitution (G/T) was found in position 173. Two nuclear genotypes (A and B) were identified in 116 individuals from 15 populations. GenBank accession numbers of the ITS2 sequences are KY314618-KY314619.
Nine haplotypes of Helianthemum recognized on basis of three chloroplast SSRs, mtrnSf-trnGr, mtrnL2-trnF, and mtrnL5-trnL3.
The cpSSR haplotypes were partitioned among the two regions: Yili Valley and western Ordos Plateau. Haplotypes H1-H4 were distributed in Yili Valley, and haplotypes H5–H9 were distributed in western Ordos Plateau. Between the two regions, there was no shared haplotypes (Figure 1B). In Yili Valley, haplotype H1 was widespread in six populations of the total nine populations; rare haplotype H2 was found in population JMC; haplotype H3 was isolated in populations BL and LK, and haplotype H4 was isolated in population BSD. In western Ordos Plateau, haplotypes H5 and H6 were found in each population; rare haplotypes, H7 and H8 were found in population KBQ, and H9 was found in population QPJ (Figure 1B). For ITS genotype, all the individuals in Yili Valley contained one genotype (A), and all the individuals in western Ordos Plateau contained the other genotype (B) (Figure 1C).
The genetic relationships among the nine haplotypes also supports the disjunct geography of the Yili Valley and Ordos Plateau. The haplotypes found in the two geographic regions are also distinct based on the haplotype network (Figure 2). These two regional lineages, haplotypes H1-H4 corresponding to Yili Valley and H5-H9 corresponding to western Ordos Plateau, were connected by at least two hypothetical haplotypes (mv3 and mv5).
Median-joining network of Helianthemum haplotypes. The blank circles indicate missing or inferred haplotypes; the circle size is proportional to haplotype frequency; haplotypes in the network showed in the same colors correspond to those in the geographical...
Genetic diversity and genetic structure
The mean different alleles number (Na) was 1.267, and effective alleles number (Ne) was 1.150. Across the entire study area, total gene diversity (hT) was 0.805 (SE 0.0632), and within-population gene diversity (hS) was 0.209 (SE 0.0679). Genetic differentiation index GST was 0.740 (SE 0.0784), and NST was 0.803 (SE 0.0617). As shown by the results of a U-test (U = 0.99, p < 0.01), NST was significantly higher than GST, suggesting a significant phylogeographical structure within Helianthemum. In Yili Valley, hT was 0.583 (SE 0.1501), hS was 0.04 (SE 0.0395), and genetic differentiation index was (GST = 0.932, NST = 0.934); in western Ordos Plateau, hT was 0.634 (SE 0.0748), hS was 0.463 (SE 0.0836), and genetic differentiation index was (GST = 0.270, NST = 0.226). AMOVA analysis showed that 79.32% (p < 0.001) of the total variation occurred among populations. When populations were grouped by geographical region, 66.81% (p < 0.001) of the total variation occurred among the regions (Table 3). In Yili Valley, 93.22% (p < 0.001) of the total variation occurred among populations; in western Ordos Plateau, 22.36% (p < 0.001) of the total variation occurred among populations. Mantel's test showed a significant correlation between genetic distance and geographical distance (r = 0.937, p < 0.001, Figure 3).
Results of analysis of molecular variance for Helianthemum based on chloroplast SSRs data.
The figure shows a significant relationship between geographic and genetic distance (r = 0.937, p < 0.001).
The PCO plot illustrates the distinct differences between regions and differences within regions. The first two axis accounted for 76.14 and 13.66% of the total variation, respectively (Figure 4). The first axis separated all the Helianthemum populations into two groups, one including populations in Yili Valley and the other including populations in western Ordos Plateau. The second axis separated all the populations in Yili Valley into three groups, one including population BSD, one including populations BL and LK, and another included the remaining populations. The PCO plots suggested a high genetic divergence between Yili Valley and western Ordos Plateau population, and also a high genetic divergence among populations within Yili Valley. The PCO plot was consistent with the structure of the NJ network (Figure 5). In the NJ network, all the populations from Yili Valley clustered into a clade (Yili Valley clade), sister to the other clade containing all the populations from western Ordos Plateau (western Ordos clade). Yili Valley clade consists of two inner clade: the first inner clade contains populations BSD, BL, and LK, and the second clade contains the remaining populations. In the first inner clade, population BSD are separate from populations BL and LK. In western Ordos clade, populations MX, QLG, and QLS from the north of the plateau cluster together, drifting apart from populations HN, KBQ, and QPJ, from the south of the plateau.
Plots of the first two coordinates based on pairwise population differentiation (Nei' s) matrix of Helianthemum.
Neighbor-Joining tree of the 15 Helianthemum populations constructed using Nei's genetic distance matrix.
Genetic divergence between Yili Valley and western Ordos Plateau
Both the nuclear and chloroplast phylogenetic analyses showed two distinct lineages in Helianthemum, distributed in Yili Valley and western Ordos Plateau (Figures 1, 2). NJ network and PCO plots also indicated the similar result (Figures 4, 5). AMOVA analysis and Mantel test both showed a high level of genetic divergence between the two regions. In addition, ploidy in H. songaricum is 2X while in H. ordosicum is 4X. Wiley (1978) stated “A single lineage of ancestral descendant populations of organisms which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fates, should be defined as species status.” The two single lineages in Helianthemum in northwestern China clarified the taxonomic confusion of the genus, supporting that populations in western Ordos Plateau should be given species rank, H. ordosicum, supposed by Zhao et al. (2000).
In early Tertiary, the terrain and climate of northwestern China were very different from the arid and mountainous conditions of today. Some species of ancient Mediterranea flora, such as Helianthemum, spread along the relic of ancient Mediterranea distribution, across the Hexi Corridor, arrived to Alxa desert (Czenda, 1977; Liu, 1995). In the late Tertiary, uplifting of the northern Tibetan Plateau caused extensive aridification in northwestern China (Zheng et al., 2003; Sun et al., 2008). During the Quaternary, glaciation began to developed in the Northern Hemisphere, and the colder climate reached its maximum at about 0.8–0.6 Ma (Williams et al., 1993). Due to the dramatic climate change, many plant species of deserts in northwestern China gradually became extinct (Liu, 1995). This climatic history suggests that ancestral Helianthemum distributed in Hexi Corridor, a passage connecting Yili Valley with western Ordos Plateau, became extinct leaving those distributed in Yili Valley and western Ordos Plateau as relics. This hypothesis is supported by the haplotype relationships shown in the median-joining network (Figure 2). The putative extinction of Helianthemum along this corridor limited gene flow between the two regions. In addition to restricted gene flow, the climate in Yili Valley likely differentiated from that of the western Ordos Plateau. Because of the Tianshan Mountains, the Yili Valley has a Central Asia climate (Liu, 1995) with hot- dry summers, and mild-humid springs and winters (Shi et al., 2005). However, climate in western Ordos Plateau is typically drier throughout the year (Walker, 1974). Helianthemum in these two regions have inhabited distinct habitats for several millennia, harboring unique populations.
Genetic diversity and genetic structure
Total genetic diversity of the two species are both moderate (H. songaricum: hT = 0.583; H. ordosicum: hT = 0.634), compared with other desert plants, such as Ammopiptanthus mongolicus (hT = 0.434), A. nunas (hT = 0.041) (Su et al., 2016), Reaumuria soongorica (hT = 0.312) (Qian et al., 2008), and are both higher than that in the previous phylogeography study of Helianthemum (H. songaricum, hT = 0.162; H. ordosicum, hT = 0.566), using two chloroplast intergenic spacers (Su et al., 2011). The inconsistency is due to higher polymorphism in the three cpSSRs than the two chloroplast spacers.
The cpSSRs data showed signs for genetic divergence in both H. songaricum and H. ordosicum. AMOVA analysis demonstrated significant genetic divergence among populations in both H. songaricum and H. ordosicum. The genetic divergence in the two species were also supported by NJ network and PCO analysis. As shown by the NJ network and PCO plots (Figures 4, 5), populations BL, LK, and BSD clustered together, apparently separated from the other populations in Yili Valley; populations HN, KBQ, and QPJ clustered together, apparently separated from the other populations in western Ordos Plateau. The significant genetic divergence among populations in the two species might be attributed to several factors. First, the seed viability is poor. In a Helianthemum flower, most ovules are unfertilized, or fertilized but with abnormal development, usually leaving only 1-3 well-developed seeds. Thus, seed production is very low (Ma et al., 2007). In addition, the seed requires a dormancy period, and with the poor water translocation due to the hard testa (Cao et al., 2000), the germination is very low (Ma et al., 2007). Second, habitats of the two species are both highly fragmented. In Yili Valley, there are several mountain ranges that subdivide Helianthemum habitat into five valleys (Zhang, 2006). The collected sites of H. songaricum are located in different secondary valleys. Similar in western Ordos Plateau, collection sites of H. ordosicum located in different valleys, along the Table Mountains. Within each species, the numerous geographic barriers isolate the populations, obstructing gene flow among them, and consequently likely decreasing genetic diversity and increasing the genetic divergence. Third, a reduced population size in the two species might also affect the population structure by increased selfing, mating among related individuals, and genetic drift. Based on congeners (Rodríguez-pérez, 2005; Aragón and Escudero, 2008), H. songaricum is likely an outcrosser but also self-compatible. Increased selfing or mating among related individuals in small population would reduce heterozygosity (Schaal and Leverich, 1996), and increased genetic drift would fix alleles randomly (Lynch et al., 1995), resulting in an alteration of population allele composition, inducing the population as an unique genetic sector (Gaudeul et al., 2000). The pattern of single haplotypes found in nearly all the populations in the Yili Valley suggests selfing is a fixture of this region. Compared to H. songaricum, H. ordosicum populations have greater population diversity (Table 3), and typically multiple haplotypes per population (Figure 1). These contrasting patterns suggest a greater degree of selfing or inbreeding in H. songaricum that could be caused by barriers to gene flow, differences in the abundance of pollinators or adaptation to a selfing life history.
Implications for conservation in Helianthemum
Habitat fragmentation is a significant threat to the survival of plant species in many terrestrial ecosystems (Young et al., 1996). Our data observed low genetic diversity in isolated small populations in Helianthemum in northwestern China. Low genetic diversity can reduce population fitness and viability, weakening the population's ability to respond to changing selection pressures, increasing the extinction vulnerability (Young et al., 1996). We suggest an effective conservation management program incorporated our genetic analysis in following manners: (i) Significant genetic divergence showed by both nuclear and chloroplast data indicates two single evolutionary lineages in Helianthemum in northwestern China. The two species should be treated, respectively, when performed a management strategy. (ii) For in situ conservation, all the natural habitat of Helianthemum populaton should be preserved by local governments. Nature reserves for H. ordosicum have been set up in western Ordos Plateau. However, in Yili Valley, conservation of H. songaricum has not been given enough attention, and we propose nature reserves for H. songaricum should be set up at once. In addition, for serious fragmentation in both species, extinct populations should be reestablished to connect remnant populations in each species, using progenies from populations with nearest geographic distance. Also, population sizes should be augmented by transplanting progenies propagated from original populations. (iii) For both species, ex situ conservation site should be established first. Seeds collection should capture all detected genetic variations to represent the genetic diversity of each species in maximum, avoiding artificially induced bottlenecks (Maunder et al., 2001). Because of genetic uniqueness of populations BSD, BL, and LK in H. songaricum, seeds collections in these populations should be deposited as separate stocks. Meanwhile, seedlings of each species should be cultured for their future restoration. In H. songaricum, crossing individuals from unique populations (BSD, BL, and LK) and the rest of the populations should be tested ex situ to prevent potential outcrossing depression.
Conceived and designed the experiments: ZS, BR. Performed the experiments: ZS. Analyzed the data: ZS, BR, LZ, and XJ. Contributed reagents/materials/analysis tools: ZS. Wrote the paper: ZS.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by grants from Natural Science Foundation of Xinjiang (2014211A073). We thank USDA Forest Service, Rocky Mountain Research Station, for the helps in the experiment.
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Despite the enormous efforts of the scientific and medical community, cancer still represents the second leading cause of death and is nearly becoming the leading one in the elderly . It is estimated that, due to demographic changes alone, in the next 15 years the number of new cancer cases will increase by 70% worldwide .
The lack of effective diagnostic tools for early detection of several tumors, the limited treatment options for patients with advanced stages of cancer, and the onset of multiple drug resistance favor poor prognosis and high mortality rates. The significant, but still unsatisfactory, improvement of survival, the severe toxicity profile, and the high costs characterizing many current anticancer therapies clearly show that a threshold in terms of clinical benefit and patients’ tolerance has been reached. Thus, the identification and development of innovative, preventive as well as therapeutic strategies to contrast cancer-associated morbidity and mortality are urgently needed.
Epidemiological, preclinical, and clinical studies have generally concluded that a diet rich in phytochemicals can reduce the risk of cancer [2,3]. Due to their pleiotropism which includes antioxidant, anti-inflammatory, and antiproliferative activities as well as modulatory effects on subcellular signaling pathways, phytochemicals from edible fruits and vegetables are recognized as an effective option to counteract cancer incidence and mortality [3,4,5]. Plants constitute a primary and large source of various chemical compounds including alkaloids, flavonoids, phenolics, tannins, tocopherols, triterpenes, and isothiocyanates. Ellagitannins (ET) are an important class of phytochemicals contained in a number of edible plants and fruits recommended by the traditional medicine of a variety of cultures, both in the developing and developed countries, to treat common health problems. ET biological and nutraceutical potential has received increasing attention over the last several decades. ET exert multiple and clinically-valuable activities , and among them the chemopreventive, anticarcinogenic, and antiproliferative activities are being receiving growing interest and attention (Figure 1).
2. Dietary Sources, Types, and Occurrence
ET and their derivatives are noticeably contained in edible seeds, nuts, and various fruits of nutritional interests. The structures of relevant ETs and of ellagic acid are shown in Figure 2. A wide variety of fresh fruits including berries, like raspberries, black raspberries, strawberries, pomegranate, longan, and dried nuts, are renowned for their ample polyphenols concentration in the form of ET . Five species of berries including raspberry, strawberry, cloudberry, rose hip, and sea buckthorn were identified by Koponen et al.,  as significant carrier of ET in a range of 1–330 mg per 100 g of fruit. Sanguiin H-6 and lambertianin C were reported from Glen Ample raspberries and Scottish-grown red raspberries, along with some trace levels of ellagic acid [7,8]. Blackberries (fruit and seeds) have been reported for a range of ET including pedunculagin, casuarictin, sanguiin H-6 (lambertianin A), and lambertianin (C and D) [9,10,11]. Pomegranate and various fractions of the fruit are known for their cancer chemopreventive properties owing to their unique phenolics composition in the form of ET, which include punicalagin, punicalin, granatin A, granatin B, tellimagrandin I, pedunculagin, corilagin, gallagic acid, ellagic acid, and casuarinin .
ET, predominately those isolated from pomegranate (e.g., punicalagin), have gained a wide popularity as preventive and therapeutic ethnopharmacological approaches for cancer treatment. However, a lot more has been added to this class of compounds from fruits other than pomegranate, including raspberries, blueberries, strawberries, muscadine grapes, and longan [7,13,14,15,16,17,18]. Major phenolic fractions recovered from longan include gallic acid, ellagic acid, and corilagin, much more concentrated in the seed segment as compared to the fruit pulp and peel . Good essential fatty acid composition of nuts and fairly high concentrations of ET and their derived fractions, such as ellagic acid and its glycosidic derivatives have been associated with the potential cardioprotective properties of nuts. Ellagic acid (free and total) has been reported in a range of 0.37–823 mg per 100 g of dried nuts . High concentrations of a variety of ET (ellagic acid, sanguiin H2 and 6, lambertianin C, castalagin/vescalagin, galloyl-bis-HHDP glucose, pedunculagin) can be found in blackberries (Rubus sp.) . Shi et al.,  identified agrimoniin as the second highest phenolic compound of strawberries.
Irrespective of the edible fractions of fruiting plants, some inedible fractions like fruit peels, bark and foliage have also been reported as good source of hydrolysable tannins including bioactive ET [4,22]. Leaves extracts of Shepherdia argentea—a deciduous shrub commonly known as silver buffaloberry—were reported as a good reserve of gluconic acid core carrying the potential anti-HIV novel ET, such as hippophaenin A, shephagenin A and shephagenin B .
3. Ellagitannins—Classification and Chemistry
Tannins are unique secondary metabolites of plant phenolics with relatively higher molecular weight (300–30,000 Da) and bear the ability to generate complexes with some macromolecules, like proteins and carbohydrates . Chemistry and nomenclature of the tannins is complicated by virtue of the frequent changes which parallel the advancement in this very specific field . Taking into account different definitions of tannins [26,27], these compounds may be referred as either galloyl esters and their derivatives (ET, gallotannins, and complex tannins), or the oligomeric and polymeric proanthocyanidins (condensed tannins). In a broader perspective, tannins may be classified most satisfactorily and unambiguously on the basis of structural configuration and/or solubility . C–C coupling of galloyl units in absence of glycosidically-linked catechin make ETs structurally different from the condensed tannins that are characterized by monomeric catechin linkages (C4–C8 or C4–C6) to generate oligomeric likewise polymeric proanthocyanidins . Gallotannins and ETs constitute a major group of tannins i.e., hydrolysable tannins that are well known for their properties to hydrolyze into hexahydroxydiphenol (HHDP) or gallic acid moieties. Gallotannins are the gallic acid derivatives carrying ≥ six gallyol groups and might further be characterized on account of one or more than one digalloyl group .
ETs (hydrolysable tannins) on their hydrolysis yield gallic acid and ellagic acid from the compounds carrying gallyol groups and HHDP groups, respectively . In vitro digestion models declare ETs to remain stable under the normal physiological condition of the stomach . However, ETs hydrolysis to free ellagic acid or their degradation may proceed in the small intestine at neutral to alkaline pH . Biologically, condensed tannins and gallotannins are thought to deliver relatively higher protein precipitation properties as compare to the ETs and hence are considered potential antinutritional compounds from the class of plants polyphenolics . Gallotannins and condensed tannins have also been reported as oxidatively least active tannins as compared to the ETs and on the same time gallotannins and condensed tannins have also been found to reduce pro-oxidant properties of ETs [33,34].
3.1. Simple Ellagitannins
ET (M.W. 300–20,000 Da) are non-nitrogenous compounds with at least two C–C coupled galloyl units with no glycosidically-bonded catechin unit [3,35]. ET are derivatives of 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG). Structurally, ET are esters of carbohydrates and or cyclitols and also include metabolic compounds derived from oxidative cleavage of either condensed or hydrolysable tannins [27,35,36]. The presence of hexahydroxydiphenol (HHDP) in a glucopyranose ring in addition to acyl units and certain HHDP metabolites such as dehydrohexahydroxydiphenol (DHHDP), valoneoyl and chebuloyl groups constitute simple ET. Tellimagrandin I and II, pedunulagin, casuarictin, and chebulagic acid originate from the specific orientation and number of acyl groups on glucose units. Variation in HHDP group originates by linking (C–C or C–O) one or more galloyl groups to HHDP unit.
Structural diversity of ET has been reported to correlate with their carrier-plants’ taxonomy and evolutionary hierarchy . More often, monomeric ET or oligomeric ET constitute the major tannic component of plant species. The monomeric compounds of the group include tellimagrandins I and II, pedunculagin, casuarictin, and potentillin. Type I hydrolysable tannins (i.e., gallotannins) carrying HHDP in stable conformation at either the 2,3 or 4,6 position on a d-glucopyranose may be referred to as a simple ET [38,39,40]. Geraniin, a type III ET, is another example of monomeric simple ET carrying a DHHDP unit linked to d-gluopyranose of HHDP unit via 1C4 conformation. Dimers of ET are generated by intermolecular oxidative coupling/condensation of simple ET.
3.2. Glycosidic Ellagitannins
Chemically, the C-glycosidic linkage of ET is established via intermolecular bonds between two monomeric units, one carrying anomeric carbon while the second one galloyl or HHDP group [3,35,41]. Most recently C-glycosidic ET including granadinin, vescalagin, methylvescalagin, castalagin, stachyurin, and casuarinin have been reported from the peel and seed fraction of camu-camu, a fruiting tree of Amazon rainforest . Woody fractions of various fruits, particularly the nuts and berries, have also been observed to hold novel C-glycosidic ET (e.g., castalagin and vescalagin). Castacrenins D and F are two other forms of C-glycosidic ET isolated from the woody fraction of Japanese chestnut and carry gallic acid/ellagic acid moieties . Treating vescalagin with Lentinula edodes generates quercusnins A and B that may be referred as fungal metabolites of C-glycosidic ET . Castacrenins D and F isolated from chestnut wood may generate oxidative metabolites, namely castacrenins E and G, by replacing pyrogallol rings of C-glycosidic ET with cyclopentone rings . Rhoipteleanins H, I, and J were reported as novel C-glycosidic ET isolated from the fruit and bark fractions of Rhoiptelea chiliantha. Structural configuration of rhoipteleanins H revealed the presence of cyclopentenone carboxy moieties that are generated by oxidation and rearrangement of C-glycosidic ET aromatic ring .
Condensate of C-glycosidic ET is another subclass of hydrolysable tannins, which includes rhoipteleanin J produced by the intermolecular condensation (C–C or C–O) of monomeric C-glycosidic ET followed by oxidation of aromatic rings of ET . Wine aged in oak wood barrels is often reported to carry oak ET, particularly the condensation products of monomeric C-glycosidic ET. The studies infer C-glycosidic ET to play a significant role in modulation of organoleptic features of wine aged in oak wood barrels .
4. Ellagitannins Pharmacokinetics
A precise knowledge of phytochemicals’ pharmacokinetics is very important to exploit their health benefits, as well as the effects of their metabolites . In vivo, ET, instead of being absorbed directly into the blood stream, are physiologically hydrolyzed to ellagic acid, which is further metabolized to biologically-active and bioavailable derivatives, i.e., urolithins, by the activity of microbiota in gastrointestinal (GI) tract [5,48]. The biological properties of ET, such as free radical scavenging, further depend on their metabolic transformation inside gut. ET recovered from pomegranate juice may be metabolically converted by gut microbiota to urolithin A, B, C, D, 8-O-methylurolithin A, 8,9-di-O-methylurolithin C, and 8,9-di-O-methylurolithin D, and some of these metabolites display higher antioxidant activity than the parental tannins themselves. For instance, urolithin C and D show an antioxidant capacity—as determined in a cell-based assay—which is 10- to 50-fold higher as compared to punicalagin, punicalin, ellagic acid, and gallic acid . This finding suggests that intestinal transformation products of ET are likely to play a central role for the antioxidant properties at least inside the GI tract. Significant differences in urolithins’ profiles in individual human subjects feed on raspberries—a renowned source of ET—have been attributed to gut microflora, whose variations on an inter-individual basis affect their capacity of hydrolyzing ET and subsequent metabolite synthesis [48,50]. The interaction of gut microbiota composition and the host endogenous excretory system is also likely to play a further role in the observed inter-individual variability . ET are highly stable under the acidic environment of stomach, and retain their composition without being hydrolyzed to simpler compounds when exposed to various gastric enzymes. Consequently the complex structure of ET impedes their gastric absorption: however, the stomach might serve as the first site of absorption of free ellagic acid and pre-hydrolyzed forms of ET.
Contrary to stomach, the neutral or alkaline environments of duodenum and small intestines, characterized by pH values ranging from 7.1 to 8.4, allow ET hydrolyzation [31,41]. In humans, ET are rapidly absorbed and metabolized, as documented by [18,52]: following ingestion of pomegranate juice (at a dose containing 25 mg of ellagic acid and 318 mg of ET), ellagic acid can be found in plasma for up to 4 h while, at later times, it is no more detectable. In contrast, another study reported that no ellagic acid could be detected in plasma during the 4 h following the juice intake , a discrepancy which has been attributed to inter-individual variability . Ellagic acid is converted by catechol-O-methyl transferase to dimethylellagic acid, which is then glucuronidated and excreted .
Finally, the microbiologically metabolized fraction of ET,