Structural and Compositional Changes in Two Marine Shell Traditional Chinese Medicines: A Comparative Analysis Pre- and Post-Calcination.

BACKGROUND: Arcae concha and Meretricis concha cyclinae concha are two marine shellfish herbs with similar composition and efficacy, which are usually calcined and used clinically. OBJECTIVE: This study investigated variations in the inorganic and organic components of Arcae concha and Meretricis concha cyclinae concha from different production regions, both Arcae concha and Meretricis concha cyclinae concha. The aim was to enhance the understanding of these two types of marine shell traditional Chinese medicine (msTCM) and provide a foundation for their future development and application. METHOD: Spectroscopic techniques, including infrared spectroscopy, X-ray spectroscopy, and X-ray fluorescence spectroscopy, were used to analyze the calcium carbonate (CaCO3) crystal and trace elements. Thermogravimetric analysis was used to investigate the decomposition process during heating. The proteins were quantified using the BCA protein assay kit. Principal component analysis (PCA) was used to classify inorganic elements in the two marine shellfish traditional Chinese medicines. RESULTS: No significant differences were found among the various production regions. The crystal structure of CaCO3 in the raw products was aragonite, but it transformed into calcite after calcination. The contents of Ca, Na, Sr, and other inorganic elements were highest. The protein content was significantly reduced after calcination. Therefore, these factors cannot accurately reflect the internal quality of TCM, rendering qualitative identification challenging. CaCO3 dissolution in the decoction of Arcae concha and Meretricis concha cyclinae concha increased after calcination, aligning with the clinical application of calcined shell TCM. PCA revealed the inorganic elements in them, indicating that the variation in trace element composition among different drugs leads to differences in their therapeutic focus, which should be considered during usage. CONCLUSIONS: This study clarifies the composition and structure changes of corrugated and clam shell before and after calcining, and it lays the foundation for the comprehensive utilization of marine traditional Chinese medicine. HIGHLIGHTS: These technical representations reveal the differences between raw materials and processed products, which will provide support for the quality control of other shellfish TCM.

Effects of hemoglobin extracted from Tegillarca granosa on the gut microbiota in iron deficiency anemia mice.

Iron deficiency anemia (IDA) is a serious threat to the health of humans around the world. Tegillarca granosa (T. granosa) is considered as an excellent source of iron due to its abundant iron-binding protein hemoglobin. This study aimed to investigate the effects of hemoglobin from T. granosa on the gut microbiota and iron bioavailability in IDA mice. Compared to normal mice, IDA mice showed reduced microbiota diversity and altered relative abundance (reduced Muribaculaceae and increased Bacteroides). After 4 weeks of administration, hemoglobin restored the dysbiosis of the intestinal microbiota induced by IDA and decreased the Firmicutes/Bacteroidota ratio and the abundance of Proteobacteria. Analysis of the hemoglobin regeneration efficiency of mice treated with hemoglobin confirmed that hemoglobin exhibited high iron bioavailability, particularly at low-dose administration, suggesting that a small amount of hemoglobin from T. granosa markedly elevated the blood hemoglobin level in mice. These findings suggested that IDA could be alleviated by administration of hemoglobin with excellent iron bioavailability, and its therapeutic mechanism may be partially attributed to the regulation of the intestinal microbiota composition and relative abundance. These results indicated that T. granosa hemoglobin may be a promising iron supplement to treat IDA and promote the utilization of aquatic-derived proteins.

Absorption of iron from Tegillarca granosa using an in vitro simulated digestion and Caco-2/HepG2 co-culture system.

BACKGROUND: Iron-deficiency anemia is one severe micronutrient malnutrition and has captured worldwide attention. This study evaluated the in vitro iron absorption of two iron-binding proteins (hemoglobin and ferritin) from Tegillarca granosa. In addition, the protein structure-iron absorption relationship and the regulatory effect of hepcidin on cellular iron absorption were explored. RESULTS: Our findings revealed that both hemoglobin and ferritin extracted from T. granosa contained abundant iron-binding sites, as evidenced by stronger peaks in amide I and II regions compared with the two proteins from humans. Less ß-sheet (27.67%) structures were found in hemoglobin compared with ferritin (36.40%), probably contributing to its greater digestibility and more release of available iron. This was confirmed by the results of Caco-2/HepG2 cell culture system that showed iron absorption of hemoglobin was 26.10-39.31% higher than that of ferritin with an iron content of 50-150 µmol L-1 . This high iron absorption of hemoglobin (117.86-174.10 ng mg-1 ) could also be due to more hepcidin produced by HepG2 cells, thereby preventing ferroportin-mediated iron efflux from Caco-2 cells. In addition, the possible risk of oxidative stress was evaluated in cells post-iron exposure. In comparison with ferrous sulfate, a common iron supplement, Caco-2 cells treated with the iron-binding proteins had a 9.50-25.73% lower level of intracellular reactive oxygen species, indicating the safety of hemoglobin and ferritin. CONCLUSION: Collectively, the data of this research would be helpful for understanding the key features and potential of developing hemoglobin and ferritin from T. granosa as novel iron supplements. © 2022 Society of Chemical Industry.

Anti-inflammatory action of ark shell (Scapharca subcrenata) protein hydrolysate in LPS-stimulated RAW264.7 murine macrophages.

Potential anti-inflammatory effects of ark shell (Scapharca subcrenata) protein hydrolysates were investigated. Ark shell protein hydrolysates were prepared using Alcalase® and pepsin and were designated ASAH and ASPH, respectively. The nitric oxide (NO) inhibitory activity of ASAH and ASPH was determined in lipopolysaccharides (LPS)-stimulated RAW264.7 murine macrophages, and the results showed that ASAH inhibited better NO inhibitory activity than ASPH. ASAH suppressed inflammatory mediator, a prostaglandin E2, secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6), and production of reactive oxygen species (ROS) dose dependently. It inhibited the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and simulated heme oxygenase-1 (HO-1) protein expression. However, the pharmacological approach revealed that pretreatment with zinc protoporphyrin ІX (ZnPP), an inhibitor of HO-1, reversed the anti-inflammatory effect of ASAH. Moreover, ASAH upregulated phosphorylation of mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK1/2, and p38 MAPK. To find out the role of MAPKs phosphorylation, MAPKs inhibitors were used, and the results showed that ASAH-mediated HO-1 protein expression and Nrf2 nuclear translocation were abolished. Taken all together, this study revealed that ASAH has a potential anti-inflammatory activity through regulation of the MAPK-dependent HO-1/Nrf2 pathway. PRACTICAL APPLICATIONS: Food-derived marine bioactive peptides, due to their pivotal role in biological activities, are gaining much attention recently. However, the anti-inflammatory activities of ark shell protein hydrolysates still remain to be investigated. This study investigated that ASAH shows potential anti-inflammatory activities through regulation of the MAPK-dependent HO-1/Nrf2 pathway in RAW264.7 murine macrophages. These findings indicated that ASAH may be used as a dietary supplement, functional food, and medicinal drug for the management of inflammation and inflammation-associated diseases.

Expression of ABCA3 transporter gene in Tegillarca granosa and its association with cadmium accumulation.

Exposure to cadmium (Cd), a heavy metal, can cause strong and toxic side effects. Cd can enter the body of organisms in several ways, leading to various pathological reactions in the body. Tegillarca granosa is a kind of bivalve shellfish favored by people in the coastal areas of China. Bivalve shellfish can easily absorb heavy metal pollutants from water bodies while filter feeding. T. granosa is considered a hyper-accumulator of Cd, and the TgABCA3 gene is highly expressed in individuals with a high content of Cd-exposed blood clam. However, it is unclear whether TgABCA3 is involved in Cd ion transport in blood clam and the molecular mechanism for the mechanism of the Cd-induced responses for maintaining cell homeostasis. In this study, the complete cDNA of the TgABCA3 gene was analyzed to provide insights into the roles of TgABCA3 in resistance against Cd in blood clam. The complete sequence of TgABCA3 showed high identity to that of TgABCA3 from other bivalves and contained some classical motifs of ATP-binding cassette transport proteins. TgABCA3 expression in different tissues was measured using real-time quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. The tissue-specific expression showed that TgABCA3 expression was highest in the gill tissue. The TgABCA3 expression in the gill tissue was silenced using the RNA interference technique. After TgABCA3 silencing, the TgABCA3 expression decreased, the Cd content increased, the oxygen consumption and ammonia excretion rates increased, and the ingestion rate decreased. These results showing that the extents of Cd accumulation and resulting toxic effects are related to expression levels and activity of TgABCA3 indicate that TgABCA3 has a protective function against Cd in the clam. This increase in Cd accumulation results in serious damage to the body, leading to the enhancement of its physiological metabolism. Therefore, the findings of the study demonstrated that TgABCA3 can participate in the transport of Cd ions in the blood clam through active transport and play a vital role in Cd detoxification.

Effect of ascorbic acid and citric acid on bioavailability of iron from Tegillarca granosa via an in vitro digestion/Caco-2 cell culture system.

Iron deficiency anaemia (IDA) has been receiving worldwide attention. Developing safe and effective iron supplements is of great significance for IDA treatment. Tegillarca granosa (T. granosa), a traditional aquaculture bivalve species in China, is considered to be an excellent source of micronutrients, but the distribution and bioavailability of these minerals have yet to be investigated. The present research was conducted to determine the contents and in vitro enzymatic digestibility of minerals in T. granosa, using beef and wheat flour as reference foods. Meanwhile, two iron-binding proteins, hemoglobin and ferritin, were extracted from T. granosa, and their structures, iron accessibility and bioavailability were investigated. Moreover, the effects of ascorbic acid (AA) and citric acid (CA), two commonly applied dietary factors, on these parameters were evaluated. Our results indicated that the mineral levels varied significantly among different food matrices, with T. granosa showing the highest contents of the tested elements. Comparison of iron absorption of meat versus wheat flour and hemoglobin versus ferritin confirmed that heme iron exhibited higher bioavailability than non-heme iron. The addition of the two organic acids notably enhanced the cellular iron uptake of T. granosa-derived proteins. This could be because AA/CA weakened hydrogen bonds within proteins and caused disordered secondary structures, thereby improving their enzymatic digestibility and releasing more soluble iron to be available for absorption. The results of this study provided a basis for the development of T. granosa-derived protein-based iron supplements, promoting the diverse utilization of marine aquatic resources.

[EFFECT OF FOOD DEPRIVATION ON TRANSFORMATION OF CAROTENOIDS IN THE BIVALVE MOLLUSC ANADARA KAGOSHIMENSIS (TOKUNAGA, 1906)].

The composition and content of carotenoids in tissues of Anadara kagoshimensis (Tokunaga, 1906) under conditions of food deprivation were analyzed over the interval of 30 days. By the end of the experiment a slight and uneven decrease of the level of total carotenoid content in tissues of foot, hepatopancreas, gills and in the sum of remaining tissues was observed. Initially ß-carotene, pectenolon, zeaxanthin, diatoxanthin, alloxanthin and esters of the latter four carotenoids were found in tissues extracts. The qualitative composition of carotenoids in the examined tissues was similar. In the course of the experiment changes were noted in both the composition of carotenoids and their content. Changes common for all tissues were shown: ß-carotene disappeared on days 3-4, over the interval of 30 days a decrease in the content of the esters of pectenolon, alloxanthin, zeaxanthin and diatoxanthin and an increase in the level of pectenolon and alloxanthin were observed. Tissue specificity was revealed: in foot and hepatopancreas tissues, at the background of decrease of the total content of esters of carotenoids, a considerable increase of pectenolon and alloxanthin concentration accompanied by an increase of diatoxanthin and zeaxanthin was observed; in gills and the sum of the remaining tissues, under similar increase in pectenolon and alloxanthin concentration, the level of diatoxanthin and zeaxanthin remained at the initial level or was even somewhat lower. The diagram of carotenod transformation is supplemented for Anadara kagoshimensis.

Zinc, copper, cadmium, and lead concentrations in water, sediment, and Anadara senilis in a tropical estuary.

Spatial and seasonal contaminations of zinc, copper, cadmium, and lead were assessed simultaneously in water, sediment, and in the bivalve Arca senilis from the Milliardaires Bay (Cote d'Ivoire) between February and October 2008. The metal load in sediments doubled from the dry season to the rainy season. On the contrary, metal concentrations in waters decreased significantly from the dry season to the rainy season. Zn and Pb concentrations in A. senilis showed similar seasonal variation with sediments. On the other hand, A. senilis regulated Cu concentrations by eliminating about twelve times the concentration accumulated during the dry season. Apparent Zn, Cu, Cd, and Pb concentration gradients were observed, but no significant differences between stations for sediment, water, and A. senilis. Concentrations in sediment increased from stations close to Abidjan Harbor towards farther stations, while concentrations in A. senilis showed a reverse gradient. The distribution gradient of A. senilis indicates pollution from local sources, but a transplant experiment is needed to better understand the observed spatial trend. Zn and Cu concentrations may pose little risk to human health and the environment, but they are the highest on the regional scale. On the contrary, Cd and Pb concentrations in A. senilis exceeded the maximum allowable limits set by the European Commission. Complementary studies including chemical speciation should be considered to provide a more accurate assessment of the risk of heavy metals to the environment.

Tegillarca granosa extract Haishengsu induces apoptosis in human hepatocellular carcinoma cell line BEL-7402 via Fas-signaling pathways.

This study was designed to investigate the apoptosis-inducing properties of Tegillarca granosa extract Haishengsu (HSS) in human hepatocellular carcinoma cell line BEL-7402. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells was determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. Apoptosis of BEL-7402 cells was demonstrated by fluorescence microscope with flow cytometry with Annexin V-FITC/PI double staining and Hoechst 33258 staining. Western blot analysis and RT-PCR were used to determine the expression levels of Fas. Expressions of caspase-8 and caspase-3 were examined by caspase activity assay and western blot analysis. HSS inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in a dose- and time-dependent manner. Our results showed HSS had positive effect on apoptosis through flow cytometry assay and fluorescence microscope. The expressions of Fas protein and mRNA were up-regulated following the treatment. Caspase-8 and caspase-3 were activated in the cells cultured with HSS. In conclusion, HSS induced apoptosis of human hepatocellular carcinoma BEL-7402 cells. The apoptosis was associated with the up-regulation of Fas and the activations of caspase-8 and caspase-3.

Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides.

The blood cockle, Tegillarca granosa, is a widely consumed clam in the Indo-Pacific region. Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. We identified the GS of T. granosa (Tg-GS) from hemocytes by 3'- and 5'-rapid amplification of cDNA ends (RACE)-PCR. The full-length cDNA consisted of 1762 bp, with a 1104-bp open reading frame encoding 367 amino acids. Sequence comparison showed that Tg-GS has homology to GS of other organisms, with 79.78% identity with GS from the Pacific oyster Crassostrea gigas, 71.98% identity with GS from the zebrafish Danio rerio, and 68.96% identity with human Homo sapiens GS. A C-beta-Grasp domain and an N-catalytic domain were identified in Tg-GS, indicating that Tg-GS should be classified as a new member of the GS family. A quantitative RT-PCR assay was used to detect mRNA expression of Tg-GS in five different tissues. Higher levels of mRNA expression of GS were detected in the tissues of hemocytes and the mantle. Up-regulation of GS by challenge with the bacteria Vibrio parahaemolyticus and with bacterial wall lipopolysaccharides showed that GS plays a role in anti-bacterial immunity. We conclude that pathogen infection significantly induces expression level of Tg- GS, and that activation of GS influences the immune response of T. granosa by increasing glutamine concentration.

Exposure-dose-response of Anadara trapezia to metal contaminated estuarine sediments: 3. Selenium spiked sediments.

Selenium enters near shore marine environments from the activities of coal-fired power stations. Although selenium is an essential element, at elevated concentrations it can cause genotoxic damage. The relationship between selenium exposure dose and response was investigated in Anadara trapezia exposed to selenium spiked sediment (5 µg/g and 20 µg/g dry mass) for 56 days. A. trapezia reached an equilibrium selenium tissue concentration (2 µg/g and 10 µg/g respectively) by day 42. Gills had significantly more selenium than the hepatopancreas and haemolymph. Between 12 and 21% of accumulated selenium in the gill and hepatopancreas was detoxified and in the metal rich granule. Most of the biologically active selenium in both tissues was in the mitochondrial fraction. Glutathione peroxidase activity and mean total glutathione concentrations for selenium exposed organisms were not significantly different to controls. The ratio of reduced to oxidised glutathione and the total antioxidant capacity were significantly reduced in selenium exposed organisms compared to control organisms. Increased selenium exposure resulted in significant increases in lipid peroxidation, lysosomal destabilisation and an increased frequency of micronuclei. A significant exposure-dose-response relationship for A. trapezia exposed to selenium enriched sediments indicates that elevated sediment selenium concentrations can increased biologically active selenium burdens and cause impairment of cellular processes and cell integrity.

Identification and characterization of a Tegillarca granosa ferritin regulated by iron ion exposure and thermal stress.

Ferritin, a conserved iron storage protein of most living organisms, plays a crucial role in iron metabolism. In this study, the ferritin gene from Tegillarca granosa (denoted as TgFER) was identified by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of TgFER was of 895bp, consisting of a 5'-UTR of 163 bp with a putative iron regulatory element (IRE), a 3'-UTR of 213 bp, and a complete open reading frame of 519 bp encoding a polypeptide with 172 amino acid residues. The predicted molecular mass of deduced amino acid of TgFER was 20.00 kDa and the theoretical pI was 4.89. The deduced amino acid of TgFER shared high identities to ferritin from abalone, oyster, clam and human. The tissue distribution of TgFER in the tissues of mantle, foot, gill, haemocytes and hepatopancreas was examined by quantitative real-time PCR (q-PCR) and mRNA transcripts of TgFER were found to be dominately expressed in haemocytes, hepatopancreas and gill and weakly in foot and mantle. The temporal expression of TgFER in haemocytes or hepatopancreases after challenged by metals ion (FeCl2 and FeCl3) exposure and thermal stress were also analyzed with q-PCR. The diverse expression patterns of TgFER were detected depending upon the types of stimulators and tissues. The ployconal antibodies generated from the recombinant product of TgFER could be specifically identified not only the recombinant product, but also the native protein from haemocytes. All these results strongly suggested that TgFER was involved in the iron metabolism and thermal stress regulation in T. granosa.

Effect of haishengsu as an adjunct therapy for patients with advanced renal cell cancer: a randomized and placebo-controlled clinical trial.

OBJECTIVE: The purpose of this study was to investigate the effect of Haishengsu, an extract from Tegillarca L. granosa, on the effects and side-effects of immunotherapy in patients with advanced renal cell cancer. METHODS: Fifty-five (55) patients with renal cell cancer were randomly divided into a Haishengsu group (n = 27, 2.4 mg, intravenously for 15 days) and a control group (n = 28). All patients were also treated with interleukin-2, interferon-alpha, and fluorouracil. RESULTS: In the Haishengsu group, the prevalence of gastrointestinal reactions to the immunotherapy was lower than in the control group (18.5% versus 64.3%, p < 0.01). In comparison with the control group, more patients from the Haishengsu group had increased food intake (74.1% versus 14.3%, p < 0.01), weight gain (77.8% versus 10.7%, p < 0.01) or an increase in Karnofsky Performance Status score (55.6% versus 17.9%, p < 0.01). The remission rate of cancer in the Haishengsu group was higher than in the control group (51.9% and 21.4%, p < 0.01). CONCLUSIONS: Addition of Haishengsu to the conventional immunotherapy is associated with an increased remission rate in patients with advanced renal cell cancer. Haishengsu was also associated with a reduced rate of gastrointestinal side-effects from the immunotherapeutic agents, and an improvement in the physical functionality of the patients.

Effect of a seashell protein Haishengsu on cell growth and expression of apoptosis genes in leukemia K562 cells.

OBJECTIVES: To investigate the effect of a seashell protein Haishengsu (HSS), an extract from a shellfish Tegillarca granosa, on cell growth and the expression of apoptosis genes in leukemia K562 cells. METHODS: Cultured K562 cells were treated with HSS at various concentrations (10-40 mg/L). The cell cycle, cell growth and the expression of apoptosis suppressor gene bcl-2 and apoptosis promoting gene bax were evaluated. RESULTS: HSS, 20mg/L, inhibited cell cycle in the G0/G1 and S phases. HSS, 20mg/L, also inhibited the growth of K562 cells over time. Expression of bcl-2 gene in the HSS 20mg/L (58.8%+/-4.7%) and HSS 40 mg/L group (26.6%+/-2.1%) were lower than in the control group (91.0+/-8.7%, P < 0.01). Expression of bax gene in the HSS 20mg/L (77.7+/-3.6%) and 40 mg/L group (90.6+/-3.7%) were higher than in the control group (10.9+/-6.6%, P < 0.01). CONCLUSION: HSS suppresses leukemia K562 cell growth by inhibiting the G0/G1 and S phases of the cell cycle. It also induces apoptosis in these leukemia cells by reducing the expression of apoptosis suppressor gene bcl-2, and increasing the expression of apoptosis promoting gene bax. Further studies are required to investigate the clinical efficacy of HSS in leukemia.

The accumulation of Zn, Se, Cd, and Pb and physiological condition of Anadara trapezia transplanted to a contamination gradient in Lake Macquarie, New South Wales, Australia.

The benthic bivalve, Anadara trapezia, was collected from a 'clean' reference site and transplanted along a suspected trace metal contamination gradient in Lake Macquarie, NSW. At monthly intervals, Zn, Se, Cd and Pb concentrations were measured in the surficial sediments and whole tissues of the cockle as well as their physiological condition (Scope for Growth). Zinc, Cd and Pb concentrations in sediments decreased together, southward, with the highest concentrations in the Cockle Bay area, suggesting that this is the main source of contamination. Zinc, Cd and Pb concentrations were near or above [ANZECC/ARMCANZ, 2000. National water quality management strategy paper 4. Australian and New Zealand Guidelines for Fresh and Marine Water Quality, Australian and New Zealand Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand. pp. 3.5.-1-3.5-10] sediment quality guidelines at Cockle Creek, Warners Bay and Koorooa Bay. Significant differences in trace metal concentrations could not be attributed to grain size or Fe concentration differences. Se concentrations were highest in fine grain Fe rich sediments of Whiteheads Lagoon, and likely to be associated with power generation operations. Trace metal concentrations did not vary significantly over time. Zinc, Cd and Pb concentrations in the tissues of A. trapezia followed a similar pattern to that of sediments. Zinc and Pb concentrations in cockles and sediments were highly correlated, indicating significant exposure-dose relationships. Selenium concentrations in the tissues of A. trapezia were higher after transplantation to the lake, however, Se concentrations were similar in all transplanted cockles, indicating that Se in contaminated sediments is not the major source of Se to organisms. There was a decline in the physiological condition of A. trapezia transplanted to Lake Macquarie after a 90-day-period with marked differences in clearance rates and respiration rates at some locations and absorption efficiencies at all locations. The mean Scope for Growth value at the most contaminated location, Cockle Bay, was markedly lower than at other locations. A significant Zn exposure-dose response relationship indicates that Zn bioaccumulation is occurring in response to sediment contamination. A significant Cd exposure-response relationship indicates that Cd may be influencing the health of cockles. Significant Pb exposure-dose, exposure-response and dose-response relationships indicate that Pb probably is affecting the health of cockles in Lake Macquarie. Therefore, Zn, Cd and Pb concentrations in sediments are likely to be affecting the health of cockles in Lake Macquarie.