The genus Balanophora J. R. Forst. & G. Forst. - Its use in traditional medicine, phytochemistry, and pharmacology: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Natural products, particularly medicinal plants, have been utilized in traditional medicine for millennia to treat various diseases. The genus Balanophora (Balanophoraceae) consists of 23 accepted species. These species are the most controversial flowering plants, with highly reduced morphologies and are found parasitizing on the roots of their host. They have been used in traditional medicine as a remedy for stomach pain, detumescence, uterine prolapse, wounds, syphilis, gonorrhea, treating injuries from falls, and other conditions. However, there is no review of this genus on its traditional uses, phytochemistry, and pharmacology. AIM: The present narrative review discusses the scientific data supporting the traditional uses of Balanophora species. The available information on its botanical properties, traditional uses, chemical contents, pharmacological activities, and toxicity was summarized to help comprehend current research and offer a foundation for future research. MATERIALS AND METHODS: The materials used in combining data on the genus Balanophora comprises online sources such as Web of Science, Google Scholar, Science Direct, and Chinese National Knowledge Infrastructure (CNKI) for Chinese-related materials. World Flora online was used in validating the scientific names of this genus while ChemBio Draw Ultra Version 22.2 software was employed in drawing the phytochemical compounds. RESULTS: Nine Balanophora species including B. harlandii, B. japonica, B. polyandra, B. fungosa, B. fungosa subsp. indica, B. laxiflora, B. abbreviata, B. tobiracola, and B. involucrata have been documented as vital sources of traditional medicines in different parts of Asia. A total of 159 secondary metabolites have been isolated and identified from the ten species of this genus comprising tannins, flavonoids, sterols, lignans, chalcones, terpenes, and phenylpropanoids. Among these compounds, tannins, lignans, terpenoids, chalcones and phenolic acids contribute to the pharmacological activities of the species in this genus with several biological activities both in vitro and in vivo such as anti-inflammatory, anti-oxidant, hypoglycemic activity, cytotoxicity, anti-microbial, melanin synthesis etc. CONCLUSION: This review summarizes the available literature on the traditional uses, pharmacological properties, and phytoconstituents of Balanophora species indicating that they contain fascinating chemical compounds with diverse biological activities. The traditional uses of the species in this genus have been confirmed by scientific data such as antimicrobial, hemostatic effect, gastroprotective activity and others. However, many species in this genus are yet unknown in terms of their botanical uses, chemical composition and biological activities. Thus, more research into the scientific connections between traditional medicinal uses and pharmacological activities, mode of action of the isolated bioactive constituents, and toxicity of other Balanophora species is needed to determine their efficacy and therapeutic potential for safe clinical application.

The Mitochondrial Genome of the Holoparasitic Plant Thonningia sanguinea Provides Insights into the Evolution of the Multichromosomal Structure.

Multichromosomal mitochondrial genome (mitogenome) structures have repeatedly evolved in many lineages of angiosperms. However, the underlying mechanism remains unclear. The mitogenomes of three genera of Balanophoraceae, namely Lophophytum, Ombrophytum, and Rhopalocnemis, have already been sequenced and assembled, all showing a highly multichromosomal structure, albeit with different genome and chromosome sizes. It is expected that characterization of additional lineages of this family may expand the knowledge of mitogenome diversity and provide insights into the evolution of the plant mitogenome structure and size. Here, we assembled and characterized the mitogenome of Thonningia sanguinea, which, together with Balanophora, forms a clade sister to the clade comprising Lophophytum, Ombrophytum, and Rhopalocnemis. The mitogenome of T. sanguinea possesses a multichromosomal structure of 18 circular chromosomes of 8.7-19.2 kb, with a total size of 246,247 bp. There are very limited shared regions and poor chromosomal correspondence between T. sanguinea and other Balanophoraceae species, suggesting frequent rearrangements and rapid sequence turnover. Numerous medium- and small-sized repeats were identified in the T. sanguinea mitogenome; however, no repeat-mediated recombination was detected, which was verified by Illumina reads mapping and PCR experiments. Intraspecific mitogenome variations in T. sanguinea are mostly insertions and deletions, some of which can lead to degradation of perfect repeats in one or two accessions. Based on the mitogenome features of T. sanguinea, we propose a mechanism to explain the evolution of a multichromosomal mitogenome from a master circle, which involves mutation in organellar DNA replication, recombination and repair genes, decrease of recombination, and repeat degradation.

Extreme plastomes in holoparasitic Balanophoraceae are not the norm.

BACKGROUND: Balanophoraceae plastomes are known for their highly condensed and re-arranged nature alongside the most extreme nucleotide compositional bias known to date, culminating in two independent reconfigurations of their genetic code. Currently, a large portion of the Balanophoraceae diversity remains unexplored, hindering, among others, evolutionary pattern recognition. Here, we explored newly sequenced plastomes of Sarcophyte sanguinea and Thonningia sanguinea. The reconstructed plastomes were analyzed using various methods of comparative genomics based on a representative taxon sampling. RESULTS: Sarcophyte, recovered sister to the other sampled Balanophoraceae s. str., has plastomes up to 50% larger than those currently published. Its gene set contains five genes lost in any other species, including matK. Five cis-spliced introns are maintained. In contrast, the Thonningia plastome is similarly reduced to published Balanophoraceae and retains only a single cis-spliced intron. Its protein-coding genes show a more biased codon usage compared to Sarcophyte, with an accumulation of in-frame TAG stop codons. Structural plastome comparison revealed multiple, previously unknown, structural rearrangements within Balanophoraceae. CONCLUSIONS: For the "minimal plastomes" of Thonningia, we propose a genetic code change identical to sister genus Balanophora. Sarcophyte however differs drastically from our current understanding on Balanophoraceae plastomes. With a less-extreme nucleotide composition, there is no evidence for an altered genetic code. Using comparative genomics, we identified a hotspot for plastome reconfiguration in Balanophoraceae. Based on previously published and newly identified structural reconfigurations, we propose an updated model of evolutionary plastome trajectories for Balanophoraceae, illustrating a much greater plastome diversity than previously known.

GC-MS/MS Quantification of EGFR Inhibitors, ß-Sitosterol, Betulinic Acid, (+) Eriodictyol, (+) Epipinoresinol, and Secoisolariciresinol, in Crude Extract and Ethyl Acetate Fraction of Thonningia sanguinea.

Medicinal plants are widely used in folk medicine to treat various diseases. Thonningia sanguinea Vahl is widespread in African traditional medicine, and exhibits antioxidant, antibacterial, antiviral, and anticancer activities. T. sanguinea is a source of phytomedicinal agents that have previously been isolated and structurally elucidated. Herein, gas chromatography combined with tandem mass spectrometry (GC-MS/MS) was used to quantify epipinoresinol, ß-sitosterol, eriodictyol, betulinic acid, and secoisolariciresinol contents in the methanolic crude extract and its ethyl acetate fraction for the first time. The ethyl acetate fraction was rich in epipinoresinol, eriodictyol, and secoisolariciresinol at concentrations of 2.3, 3.9, and 2.4 mg/g of dry extract, respectively. The binding interactions of these compounds with the epidermal growth factor receptor (EGFR) were computed using a molecular docking study. The results revealed that the highest binding affinities for the EGFR signaling pathway were attributed to eriodictyol and secoisolariciresinol, with good binding energies of -19.93 and -16.63 Kcal/mol, respectively. These compounds formed good interactions with the key amino acid Met 769 as the co-crystallized ligand. So, the ethyl acetate fraction of T. sanguinea is a promising adjuvant therapy in cancer treatments.

Cytonuclear coevolution in a holoparasitic plant with highly disparate organellar genomes.

KEY MESSAGE: Contrasting substitution rates in the organellar genomes of Lophophytum agree with the DNA repair, replication, and recombination gene content. Plastid and nuclear genes whose products form multisubunit complexes co-evolve. The organellar genomes of the holoparasitic plant Lophophytum (Balanophoraceae) show disparate evolution. In the plastid, the genome has been severely reduced and presents a > 85% AT content, while in the mitochondria most protein-coding genes have been replaced by homologs acquired by horizontal gene transfer (HGT) from their hosts (Fabaceae). Both genomes carry genes whose products form multisubunit complexes with those of nuclear genes, creating a possible hotspot of cytonuclear coevolution. In this study, we assessed the evolutionary rates of plastid, mitochondrial and nuclear genes, and their impact on cytonuclear evolution of genes involved in multisubunit complexes related to lipid biosynthesis and proteolysis in the plastid and those in charge of the oxidative phosphorylation in the mitochondria. Genes from the plastid and the mitochondria (both native and foreign) of Lophophytum showed extremely high and ordinary substitution rates, respectively. These results agree with the biased loss of plastid-targeted proteins involved in angiosperm organellar repair, replication, and recombination machinery. Consistent with the high rate of evolution of plastid genes, nuclear-encoded subunits of plastid complexes showed disproportionate increases in non-synonymous substitution rates, while those of the mitochondrial complexes did not show different rates than the control (i.e. non-organellar nuclear genes). Moreover, the increases in the nuclear-encoded subunits of plastid complexes were positively correlated with the level of physical interaction they possess with the plastid-encoded ones. Overall, these results suggest that a structurally-mediated compensatory factor may be driving plastid-nuclear coevolution in Lophophytum, and that mito-nuclear coevolution was not altered by HGT.

An UHPLC/LC-MS illustrated the dynamic profiling of balanophorin B, gallic acid, and 4-hydroxycinnamic acid in rat as 3 molecular entities from Balanophora simaoensis.

An UHPLC/LC-MS was founded to detect balanophorin B (B), gallic acid (GA), 4-hydroxycinnamic acid (HC), and their in vivo profiling in rats, after oral administration of the ethanol extract of Balanophora simaoensis S. Y. Chang et Tam. The in vivo dynamic existence of 3 molecular entities in rats and the multistep biotransformation of GA were elucidated by their sensitive mass spectrometry response after efficient UHPLC and/or HPLC separation, through analyzing the bio-samples of rat plasma, bile, liver, kidneys, and excreta. The method was validated with satisfactory calibration curves having correlation coefficients r from 0.996 to 0.999 for concentration scaled from 0.100 nM to 0.100 µM, internal standard normalized matrix factors ranged from 0.923 to 0.993, sextuplicate recoveries valued from 95.0% to 103.6%, as well as accuracy and precision varied from 95.6% to 103.7%. The content of B, GA, and HC in the whole herb was of 4.66, 63.5, and 10.4 µmol/kg in dry weight, respectively. The Cmax for B, GA, and HC in rat systemic circulation was of 76.0 nM, 2.30 µM, and 51.0 µM, with tmax at 3, 2, and 2 h, respectively. B and GA stayed in rat liver over 4 hs to present a material base for the pharmacology and pharmacodynamics of the whole herb. The biotransformation of GA indicated a complicated scheme in rats. As a final metabolite from GA with total biotransformation conversion over 20%, 4-hydroxybenzaldehyde resourced from two steps of dehydroxylation and one step of reduction of GA, but not concerned with HC.

Native and foreign mitochondrial and nuclear encoded proteins conform the OXPHOS complexes of a holoparasitic plant.

The intimate contact between the holoparasitic plant Lophophytum mirabile (Balanophoraceae) and its host plant (Fabaceae) facilitates the exchange of genetic information, increasing the frequency of horizontal gene transfer (HGT). Lophophytum stands out because it acquired a large number of mitochondrial genes (greater than 20) from its legume host that replaced the majority of the native homologs. These foreign genes code for proteins that form multisubunit enzyme complexes, such as those in the oxidative phosphorylation system (OXPHOS) and cytochrome c maturation (ccm) system, together with dozens of nuclear-encoded subunits. However, the existence and the origin of the nuclear subunits that form the major part of the OXPHOS and ccm system in Lophophytum remain unknown. It was proposed that nuclear-encoding genes whose products interact with foreign mitochondrial proteins are also foreign, minimizing the incompatibilities that could arise in the assembly and functioning of these multiprotein complexes. We identified a nearly complete set of OXPHOS and ccm system subunits evolving under selective constraints in the transcriptome of Lophophytum, indicating that OXPHOS is functional and resembles that of free-living angiosperms. Maximum Likelihood phylogenetic analyses revealed a single case of HGT in the nuclear genes, which results in mosaic OXPHOS and ccm system in Lophophytum. These observations raise new questions about the evolution and physiology of this parasitic plant. A putative case of cooperation between two foreign (one mitochondrial and one nuclear) genes is presented.

ITS1 Barcode and Phytochemical Analysis by Gas Chromatography-Mass Spectrometry of Corynaea crassa Hook. f (Balanophoraceae) from Ecuador and Peru.

The use of medicinal plants is the basis of traditional healthcare. Recently, the use of herbal medicine has been increasing among consumers due to availability, economy, and less side effect. For instance, the hemiparasite plant Corynaea crassa has medicinal properties and could be found in some regions of America, from Costa Rica to Bolivia. Phytochemical and genetic characterization of medicinal plants is needed for proper identification of metabolites responsible for medicinal properties and for genotyping, respectively. Moreover, characterization of medicinal plants through the use of DNA barcodes is an important tool for phylogenetic analysis and identification of species; furthermore, complemented with phytochemical analysis, both are useful for identification of plant species and quality control of medicinal products. The objective of this study was to analyze the species of C. crassa collected in Ecuador and Peru from the phylogenetic and phytochemical point of view. Polymerase chain reaction (PCR) was performed for amplification of the internal transcribed spacer 1 (ITS1) region after DNA extraction of samples of C. crassa. Blast analysis was performed in the GenBank database with the ITS1 sequences obtained from two accessions of C. crassa from Ecuador (GenBank accession numbers OM471920 and OM471919 for isolates CIBE-17 and CIBE-18, respectively) and three from Peru (GenBank accession numbers OM471921, OM471922, and OM471923 for isolates CIBE-13, CIBE-14, and CIBE-15, respectively). The accessions available in the GenBank were used for phylogenetic analysis. For the phytochemical analysis, hydroalcoholic extracts were obtained by maceration using 80% ethanol as solvent, followed by a derivatization process and analysis by gas chromatography-mass spectrometry. Based on the phylogenetic analysis of the C. crassa samples, the ITS1 sequence could be used to differentiate C. crassa of different locations. The samples of C. crassa from Ecuador and Peru are more similar between them than with other clades including Helosis spp. The phytochemical study revealed differences in the presence and relative abundance of some metabolites; mainly eugenol, 1,4-lactone arabinonic acid, dimethoxyrabelomycin and azelaic acid, which are reported for the first time for the species under study and the genus Corynaea. These results are the first findings on the combined analysis using genetic and phytochemical analysis for C. crassa, which could be used as a useful tool for quality control of the C. crassa species in medicinal products.

The minicircular and extremely heteroplasmic mitogenome of the holoparasitic plant Rhopalocnemis phalloides.

The plastid and nuclear genomes of parasitic plants exhibit deeply altered architectures,1-13 whereas the few examined mitogenomes range from deeply altered to conventional.14-20 To provide further insight on mitogenome evolution in parasitic plants, we report the highly modified mitogenome of Rhopalocnemis phalloides, a holoparasite in Balanophoraceae. Its mitogenome is uniquely arranged in 21 minicircular chromosomes that vary in size from 4,949 to 7,861 bp, with a total length of only 130,713 bp. All chromosomes share an identical 896 bp conserved region, with a large stem-loop that acts as the origin of replication, flanked on each side by hypervariable and semi-conserved regions. Similar minicircular structures with shared and unique regions have been observed in parasitic animals and free-living protists,21-24 suggesting convergent structural evolution. Southern blots confirm both the minicircular structure and the replication origin of the mitochondrial chromosomes. PacBio reads provide evidence for chromosome recombination and rolling-circle replication for the R. phalloides mitogenome. Despite its small size, the mitogenome harbors a typical set of genes and introns within the unique regions of each chromosome, yet introns are the smallest among seed plants and ferns. The mitogenome also exhibits extreme heteroplasmy, predominantly involving short indels and more complex variants, many of which cause potential loss-of-function mutations for some gene copies. All heteroplasmic variants are transcribed, and functional and nonfunctional protein-coding variants are spliced and RNA edited. Our findings offer a unique perspective into how mitogenomes of parasitic plants can be deeply altered and shed light on plant mitogenome replication.

Parasitic plant, from inside out: endophytic development in Lathrophytum peckoltii (Balanophoraceae) in host liana roots from tribe Paullineae (Sapindaceae).

BACKGROUND AND AIMS: Balanophoraceae is one of the most bizarre and biologically interesting plant clades. It groups species with peculiar features that offers an opportunity for investigating several aspects of parasite plant development and morphogenesis. We analysed the development and the mature vegetative body of Lathrophytum peckoltii Eichler, focusing on the formation of the host-parasite interface. Additionally, we analysed how this parasitic interaction causes modifications to the anatomy of Paullinia uloptera Radlk and Serjania clematidifolia Cambess host roots. METHODS: Vegetative bodies of the parasite at different developmental stages were collected while infesting the roots of Sapindaceae vines. Non-parasitized host roots were also collected for comparison. Light, epifluorescence, confocal and scanning electron microscopy were used for the analysis. KEY RESULTS: The initial cells of the vegetative axis divide repeatedly, originating a parenchymatous matrix, which occupies the space from the cortex to the vascular cylinder of the host's root. In the peripheral layers of the matrix, located near the xylem of the host's roots, a few cells initiate the process of wall lignification, originating the parasitic bundle. The vascular cambium of the host's root changes the division plane and becomes composed of fusiform initials, forming the vascular bundle. The vegetative axis presents a dermal tissue similar to a phellem, a parenchymatous matrix and a vascular system with different origins. CONCLUSION: The parasite reproduces by endophytic development, in a manner similar to that observed for endoparasites. The strategy of late cell differentiation could aid the parasite in avoiding early detection and triggering of defence responses by the host. This development causes changes to the host root cambial activity, leading to the establishment of direct, vessel to vessel connection between host and parasite. We associate these changes with the cambium modularity and an influx of parasite-derived hormones into the host cambium.

Phytochemical composition and biological screening of two Lophophytum species / Composición fitoquímica y cribado biológico de dos especies de Lophophytum

Lophophytum species are holoparasites that grow on tree roots. The objectives of the work were to explore the chemical composition of the tubers of two Lophophytum species and to analyze the antioxidant, anti-inflammatory and antilithiatic activity of their extracts using in vitro methods. The chemical composition was determined by histochemical, phytochemical and TLC tests. In addition, the profile of phenolic compounds was determined by HPLC-MS. The presence of secondary metabolites of recognized activity was demonstrated. The results of the HPLC-MS/MS allowed the tentative identification of catechin, luteolin and glycosides of eriodictyol, naringenin and luteolin in the extract of Lophophytum leandriand eriodictyol, naringenin, luteolin and their glycosylated derivatives in Lophophytum mirabile. The extracts showed promising antioxidant (DPPH, ABTS and ß-carotene-linoleic acid), anti-inflammatory (inhibition of 5-LOX) and anti-urolytic (by bioautographic TLC) activity. It is noteworthy that these are the first results of the phytochemical composition and biological activity of L. mirabile. However, in vivo studies are required to corroborate these activities.

Las especies de Lophophytumson holoparásitas que crecen en raíces de árboles. Los objetivos del trabajo fueron explorar la composición química del túber de dos especies de Lophophytum y analizar la actividad antioxidante, antiinflamatoria y antilitiásica de sus extractos usando métodos in vitro. La composición química se determinó mediante pruebas histoquímicas, fitoquímicas y por TLC. Además, se determinó el perfil de compuestos fenólicos por HPLC-MS/MS. Se demostró presencia de metabolitos secundarios de reconocida actividad. Los resultados del HPLC-MS/MS permitieron identificar tentativamente catequina, luteolina y glucósidos de eriodictiol, naringenina y luteolina en el extracto de Lophophytum leandriy eriodictiol, naringenina, luteolina y sus derivados glicosilados en Lophophytum mirabile. Los extractos mostraron prometedora actividad antioxidante (DPPH, ABTS y ß-caroteno-ácido linoleico), antiinflammatoria (inhibición de la 5-LOX) y antiurolitiásica (por TLC bioautográfica). Es de destacar que estos son los primeros resultados de composición fitoquímica y actividad biológica de L. mirabile. Sin embargo, se requieren estudios in vivo para corroborar dichas actividades.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes.

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.

Polysaccharides extracted from balanophora polyandra Griff (BPP) ameliorate renal Fibrosis and EMT via inhibiting the Hedgehog pathway.

Renal interstitial fibrosis (RIF) is a crucial pathological change leading to chronic kidney disease (CKD). Currently, no effective medicines have been available for treating it. In our research, we examined the effects of polysaccharides extracted from Balanophora polyandra Griff (BPPs) on kidney fibrosis and epithelial to mesenchymal transition (EMT) in vivo and in vitro, aiming to explore the underlying mechanisms. By using the mice with unilateral urethral obstruction (UUO) as experimental subjects, we examined the medicinal values of BPPs on alleviating RIF. The effects of BPPs were evaluated by examining the histological staining and relative mRNA and protein expression levels of the related genes. The possible underlying mechanisms were further explored with human normal renal proximal tubular epithelia (HK-2 cells) as in vitro model. In UUO mice, BPP treatment could significantly alleviate interstitial fibrosis through reducing the components (Collagens I, III and IV) of extracellular matrix (ECM), and reducing the activation of fibroblasts producing these components, as revealed by inhibiting the hallmarks (fibronectin and α-SMA) of fibroblast activation. Furthermore, BPP administration increased the expression levels of matrix metalloproteinases (MMPs) and declined those of tissue inhibitors of metalloproteinases (TIMPs). BPPs markedly ameliorated EMT in both the kidneys of UUO mice and TGF-ß1 treated HK-2 cells. Moreover, BPP treatment decreased the expression levels of several transcriptional factors involved in regulating E-cadherin expression, including snail, twist and ZEB1. Additionally, the Hedgehog pathway was found to be closely correlated with renal fibrosis and EMT. Altogether, our results clearly demonstrated that BPP treatment effectively inhibited the Hedgehog pathway both in renal tissues of UUO mice and TGF-ß1-treated HK-2 cells. Thus, BPPs ameliorated RIF and EMT in vivo and in vitro via suppressing Hedgehog signalling pathway.

New butenolides with anti-inflammatory activity from Balanophora fungosa.

Two new butenolides, balanolides A (1) and B (1), were isolated from the aerial parts of the parasitic plant Balanophora fungosa. Their structures were established by comprehensive spectroscopic analysis and quantum chemical ECD calculation. The new butenolides were evaluated for their inhibitory effects against nitric oxide (NO) production in LPS-induced RAW 264.7 macrophage cells. Compounds 1 and 2 displayed moderate anti-inflammatory activity with IC50values of 11.8 and 12.9 µM, respectively.

Balanophorin B inhibited glycolysis with the involvement of HIF-1α.

AIMS: Cancer cells exhibit a metabolic change called aerobic glycolysis compared with normal cells. Balanophorin B is a terpenoid ingredient reported from the genus Balanophora. In this research, we studied the effect of balanophorin B on glycolysis of HepG2 cells and Huh-7 cells under hypoxia. MAIN METHODS: The Warburg effect was monitored by assessing glucose uptake, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Key enzymes in the glycolytic pathway and HIF-1α protein expression and degradation were analyzed by real-time PCR and western blotting. The anti-cancer effect of balanophorin B in vivo was also investigated. KEY FINDINGS: Balanophorin B inhibited the proliferation, glucose uptake, and ECAR in both HepG2 cells and Huh-7 cells. In addition, balanophorin B inhibited the protein level of HIF-1α and its downstream targets LDHA and HKII under hypoxia, whereas HIF-1α mRNA level did not change after balanophorin B treatment. The HIF-1α plasmid reversed the inhibition of balanophorin B on glycolysis, and the proteasome inhibitor MG132 attenuated the effect of balanophorin B on HIF-1α protein expression, suggesting that balanophorin B might post-transcriptionally affect HIF-1α. Moreover, balanophorin B increased the expression of VHL and PHD2. HIF-1α siRNA also greatly attenuated the inhibitory effect of balanophorin B on HepG2 cells glucose uptake. Balanophorin B significantly inhibited tumor growth in vivo, without causing obvious toxicity to mice. SIGNIFICANCE: These data suggest that balanophorin B inhibits glycolysis probably via an HIF-1α-dependent pathway, and the ubiquitin-proteasome pathway was greatly involved in the induction of balanophorin B on HIF-1α degradation.

Phenolics from the Bolivian highlands food plant Ombrophytum subterraneum (Aspl.) B. Hansen (Balanophoraceae): Antioxidant and α-glucosidase inhibitory activity.

The Balanophoraceae Ombrophytum subterraneum is an endemic highland food plant occurring in Bolivia, northern Argentina and Chile. The upper part of this parasitic plant is eaten fresh. The aim of this work was to characterize the compounds occurring in the edible part and to assess any differences between the inflorescence and tuber. Ethanol extracts of the different plant parts were analyzed for antioxidant activity by the ORAC, TEAC, FRAP, CUPRAC and DPPH methods as well as for the effect on enzymes related to metabolic syndrome (α-amylase, α-glucosidase and pancreatic lipase). The main constituents were isolated by a combination of gel permeation in Sephadex LH-20 and countercurrent chromatography (CCC). Five compounds were isolated and fully identified by spectroscopic and spectrometric means. The new 3',5,5',7-tetrahydroxyflavanone 7-O-ß-D-1 â†’ 6 diglucoside was isolated for the first time. HPLC-ESI-MS/MS analysis allowed the identification of 19 compounds, including flavanones, flavanols, flavonols, dehydroflavonols and lignans, mainly as glycosides. A strong inhibition towards α-glucosidase was observed for the edible parts (IC50: 1.46 µg/mL) as well as for the tuber and inflorescence (IC50: 1.56 µg/mL and 0.87 µg/mL, respectively). A significant correlation was established between the total phenolic and the antioxidant capacity of the extracts as well as with the content of the new flavanone diglucoside. This is the first comprehensive report on the naturally occurring antioxidants and enzyme inhibitors from this native highland food resource.

Anti-neuraminidase activity of chemical constituents of Balanophora involucrata.

Balanophora involucrata J. D. Hooker has been known to possess potential anti-inflammatory and antibacterial activities; however, its antiviral activity has not been evaluated so far. In order to find new neuraminidase inhibitors (NAIs), the neuraminidase (NA) inhibition activity of different B. involucrata extracts was evaluated. In this study, an in vitro NA inhibition assay was performed to identify which extract of B. involucrata exhibits (maximal) inhibitory activity against NA. Ultra high performance liquid chromatography/quadrupole time-of-flight-tandem mass spectroscopy (MS/MS) and molecular docking techniques were used to identify the specific compounds responsible for the anti-influenza activity of the extract, and to explore the potential natural NAIs. The ethyl acetate extract of B. involucrata exhibited significant inhibitory activity against NA with 50% inhibitory concentration (IC50 ) value of 159.5 µg/mL. Twenty compounds were identified according to the MS/MS spectra; among them two compounds (quercitrin and phloridzin) showed obvious inhibitory activity against NA, with IC50 of 311.76 and 347.32 µmol/L, respectively. This study suggested that B. involucrata can be a potential natural source of NAIs and may be useful in the fight against ferocious influenza viruses.

Polysaccharides derived from Balanophora polyandra significantly suppressed the proliferation of ovarian cancer cells through P53-mediated pathway.

Ovarian cancer (OC) is ranked the first among the cancers threatening women's health. It attracts tremendous attention of cancer researchers because of its extremely high mortality rate. Recent studies have indicated that traditional herbal medicines (THMs) can play a pivotal role in cancer prevention and treatment. THMs are gaining popularity as a source of anti-cancer agents. The plant of Balanophora polyandra, which has been used as a traditional herbal medicine, has been known for exhibiting potential haemostatic, analgesic, anti-inflammatory and anti-cancer properties. However, few studies on inhibitory effect of B. polyandra on OC have been performed. In the present study, we found that B. polyandra polysaccharides (BPP) induced cell cycle arrest at S phase, triggered apoptosis and inhibited migration and invasion of OC cells. Furthermore, we also found that there was a potential and close relationship between BPP and P53-mediated pathway. Overall, these findings suggest that BPP can be a potential therapeutic agent for the treatment of OC.

Balanophora polyandra Griff. prevents dextran sulfate sodium-induced murine experimental colitis via the regulation of NF-κB and NLRP3 inflammasome.

Balanophora polyandra Griff. (B. polyandra) is a folk medicine used as an antipyretic, antidote, haemostatic, dressing and haematic tonic, for the treatment of gonorrhea, syphilis, wounds, and the bleeding of the alimentary tract by the local people in China. This study was designed to investigate the effects of B. polyandra on dextran sulfate sodium (DSS)-treated colitis mice in vivo and lipopolysaccharide (LPS)-induced RAW 264.7 macrophages in vitro. Mice were induced with B. polyandra total extract (BPE, 250 and 1000 mg kg-1) and B. polyandra polysaccharides (BPP, 100 and 400 mg kg-1) for 22 days and treated with 3.5% DSS in their drinking water for the last 7 days and the LPS-induced RAW264.7 macrophages were treated with BPE (100 µg ml-1) and BPP (100 µg ml-1). Mice treated with DSS developed severe mucosal colitis, with a marked distortion and crypt loss of colonic surface epithelium and a colonic shortening. B. polyandra significantly inhibited colonic shortening and reduced the severity of colitis in the colon and lowered the colonic inflammation score (p < 0.05) and decreased the expression of interleukin (IL)-1ß, tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), and anti-serum amyloid A3 (SAA3) as well as the pro-inflammatory chemokine C-X-C motif chemokine 10 (CXCL10). B. polyandra also significantly suppressed the activation of nucleotide-binding domain like receptor protein 3 (NLRP3) inflammasome and the nuclear factor kB (NF-κB). These results suggest that dietary intake of B. polyandra ameliorates colitis. Such activities of B. polyandra in humans remain to be investigated.

Multichromosomal structure and foreign tracts in the Ombrophytum subterraneum (Balanophoraceae) mitochondrial genome.

Horizontal gene transfer (HGT) is frequent in parasitic plant mitochondria as a result of vascular connections established in host-parasite relationships. Recent studies of the holoparasitic plant Lophophytum mirabile (Balanophoraceae) revealed the unprecedented acquisition of a large amount of mitochondrial sequences from its legume host. We focused on a close relative, the generalist holoparasite Ombrophytum subterraneum, to examine the incidence of HGT events in the mitochondrial genome (mtDNA). The mtDNA of O. subterraneum assembles into 54 circular chromosomes, only 34 of which contain the 51 full-length coding regions. Numerous foreign tracts (totaling almost 100 kb, ~ 14% of the mtDNA), including 12 intact genes, were acquired by HGT from the Asteraceae hosts. Nine chromosomes concentrate most of those regions and eight are almost entirely foreign. Native homologs of each foreign gene coexist in the mtDNA and are potentially functional. A large proportion of shorter regions were related to the Fabaceae (a total of ~ 110 kb, 15.4%), some of which were shared with L. mirabile. We also found evidence of foreign sequences donated by angiosperm lineages not reported as hosts (Apocynaceae, Euphorbiaceae, Lamiaceae, and Malvales). We propose an evolutionary hypothesis that involves ancient transfers from legume hosts in the common ancestor of Ombrophytum and Lophophytum followed by more recent transfer events in L. mirabile. Besides, the O. subterraneum mtDNA was also subjected to additional HGT events from diverse angiosperm lineages, including large and recent transfers from the Asteraceae, and also from Lamiaceae.