Long Goals and Objectives:

Investigation of bioactive compounds from marine medicinal materials in the South Central Coast of Vietnam (from Khanh Hoa to Binh Thuan) for evaluating the potential usage of marine medicinal materials in people’s health-care. Training the researchers of Vietnam to reach the research level in Asian countries regarding marine bioactive compounds. Improvement of the publications on books, patents (invention or utility solution), national journals, and international journals of marine research in Vietnam.

Recent Goals and Objectives:

+ Explore of bioactive compounds and astablishment of processes for isolating them from selected marine organisms in research areas.

+ Production of functional food for people’s health-care.

+ Evaluation of potential application of collected marine species.

+ The 10 sponge species including Aaptos aaptos (Schmidt, 1864), Amorphinopsis fenestrata (Ridley, 1884), Clathria (Thalysias) reinwardti (Vosmaer, 1880), Halichondria panicea (Pallas, 1766), Haliclona (Gellius) cymaeformis (Esper, 1806), Haliclona (Halichoclona) vansoesti (de Weerdt, de Kluijver & Gomez, 1999), Hippospongia fistulosa (Lendenfeld, 1889), Ianthella basta (Pallas, 1766), Rhabdastrella globostellata (Carter, 1883), và Xestospongea testudinaria (Lamark, 1815) were collected and archived their voucher specimens. Additonally, a sponge species, Rhabdastrella globostellata (Carter, 1883) was taxonomically identified by analysis of their DNA indicator and comparison with global gen bank.

+ From 10 sponge species, 84 different compounds were isolated and identified by moder spectrosopic methods such as HR-ESI-MS, 1D- and 2D-NMR, Circular dichroism spectroscopy and theoretical calculation of the spectral data using Spartan18 and Gaussian16 softwares. Among 84 obtained compounds, there were 23 new compounds comprising 5 novel compounds. The isolated compounds were categorized in merosesquiterpene, diterpene, sesterterpene, isomalabaricane-type triterpene and its 

analogs, acetylenic fatty acid and their lactone containing bromo atoms, phenolic, and aklaloids in terms of aaptamine alkaloid and indole alkaloid. Some compounds belonging phenolics, nucleosides and indole alkaloids were found in several sponge species.

In partitucally, 16 compounds (4 new and 3 novel compounds) isolated from Rhabdastrella globostellata, 8 compounds isolated from Amorphinopsis fenestrata, 16 compounds isolated from Hippospongia fistulosa (8 new compounds), 8 compounds isolated from Haliclona (Gellius) cymaeformis, 8 compounds isolated from Haliclona (Halichoclona) vansoesti, 8 compounds (1 new) isolated from Halichondria panicea, 10 compounds isolated from Xestospongia testudinaria, 13 compounds (2 new and 2 novel compounds) isolated from Aaptos aaptos, 11 compounds (1 new) isolated from Clathria (Thalysias) reinwardti, 9 compounds isolated from Ianthella basta. 

+ The isolated compounds were evaluated their cytotoxic activity towards LU-1, MCF-7, HepG2, SK-Mel2, and HEK-293A cell lines. Thirteen compounds (RG2, RG4, and RG8 isolated from Rhabdastrella globostellata; HP1 and HP2 isolated from Halichondria panicea; AA3, AA4, AA6, and AA7 isolated from Aaptos aaptos; CR5 isolated from Clathria (Thalysias) reinwardti; IB2, IB5, and IB6 isolated from Ianthella basta) exhibited potent cytotoxic activity with their IC50 lower than 20 µM. Especially, compounds IB2 and IB5 showed IC50 values (around 1 µM) lower than positive control ellipticine. CR5 showed significant cytotoxic against cancerous cell lines meanwhile less cytotoxic effects towards normall cells.

+ Compounds RG1, RG2, RG8, CR5, IB2, and IB5 were selected for quantitative analysis their contents in the methanol extract of the sponges. Results indicated that compounds IB2 and IB5 were high contents (1.11% and 0.64% w/w, respectively) in the methanol extracts of the sponge Ianthella basta which could be isolated in the large amounts.

+ 30 coral and echinoderm samples were collected in South Central region of Vietnam. These samples in coastal areas of Van Phong bay - Khanh Hoa, Ninh Thuan, Binh Thuan, belong to 26 species including 2 sea cucumbers, 7 starfish, 2 crinoids, 2 sea urchins, and 13 corals.

+ 07 corals and echinoderms were selected for studying chemical constituents and biological activities including 01 crinoid, 01 sea cucumber, 01 starfish, and 04 corals.

+ Investigated the chemical constituents of the 07 sellected corals and echinoderms and isolated, structurally elucidated 72 compounds including 23 from crinoid, 07 from sea cucumber, 05 from starfish, and 37 from soft corals.

+ Evaluated the biological activities of pure compounds and found that triterpene saponins exhibited potent cytotoxicity against all five tested human cancer cell lines while some naphthopyrones showed significant and sellective effects on the SK-Mel-2 cell line.

- Collection of 126 marine samples at two areas (Nha Trang–Khanh Hoa, Ninh Thuan – Binh Thuan) of the South Central region of Vietnam’s Sea.

- From 126 samples collected in the South Central Coast of Vietnam, 186 strains were isolated, including: 136 actinomycetes strains and 50 fungi strains (including 50 actinomycetes and 20 fungi strains collected in Nha Trang - Khanh Hoa; 86 actinomycetes and 30 fungi strains collected in Ninh Thuan - Binh Thuan).

- All isolated strains  which were fermented at small scale screened for their antimicrobial and antituberculosis activities. Accordingly, 15 strains which exhibited highest activities were selected for their taxonomic identification, fermentation in large scale and secondary metabolites analysis.

- All extracts of the culture of 15 bacteria strains were fractionated by chromatography methods that led to the isolation and characterization of 156 compounds, including 5 new secondary metabolites (G666-1, G666-2, G666-3, G631-2 và M893-1).

- All the isolates were evaluated for their antimicrobial and antituberculosis activity. The results showed that 37 compounds had antimicrobial activity. 7 compounds (M839-5, M881-2, M881-9, M839-1, M839-3, G769-7 and G769-8) had antimicrobial activity against 6 out of 7 tested strains, 9 compounds (G631-4, G631-7, M893-1, M893-4, M893-6, G657-1, G657-4, M881-4 and M881-10)  had antimicrobial activity against 5 out of 7 tested strains, 3 compounds (G666-1, G631-6 and G631-9) had antimicrobial activity against 4 out of 7 tested strains. For the antituberculosis activity, compound M881-2 moderately inhibited against H37Rv.

+ The project has collected 125 samples of marine organisms, including 34 samples of Coral, 21 samples of Mollusks, 19 samples of Seaweed, 32 samples of Echinoderm, 19 samples of Sponges in the coastal areaof Khanh Hoa - Binh Thuan.

+ Conduct lipid research on all collected samples:obtain data on total lipid content, composition and content of fatty acids in total lipids, composition and content of lipid classes in total lipids, composition and the content of fatty acids in the polar and non-polar lipid fractions of 30 samples;Analyze the molecular species of several classes of polar lipids in 5 marine samples. Conducted biological assays of all 125 total lipid extracts.Successfully built a database of lipid for 125 marine organisms.

+ The 1-O-alkyl-2-DHA-phosphatidylcholine mixture was prepared from tuna liver raw materials including 4 stages, using chromatography and synthesis techniques:Isolate PC and lyso PC from total lipid mixture using column chromatography,prepare 1-O-alkyl-2-OH-phosphatidylcholine mixture by hydrolysis reaction of PC + Lyso PC in alkaline environment, and prepare 1-O -alkyl-2-DHA-phosphatidylcholine by reaction of 1-O-alkyl-2-OH-phosphatidylcholine with DHA using DCC/DMAP catalyst. The products were analyzed by MS spectra.

+ Studied and evaluated the nutritional and chemical indicators of black seahorses raised in the South Central Coast (Khanh Hoa).From black seahorse raw materials, a technological process for hydrolyzing black seahorse has been built using the method of using ultrasound combined with enzymes to create a hydrolyzed seahorse powder product containing all the active ingredients.

-16 sea slug samples (belonging to 14 species of 10 genera), and 12 samples of sea snails (belonging to 11 species of 10 genera) were collected in the waters of Van Phong, Nha Trang - Khanh Hoa, Hon Cau, Ca Na - Ninh Thuan, Phu Quy - Binh Thuan. Their scientific names were identified.

- Chemical constituents of 3 sea slug species and 4 sea snail species were investigated. 86 compounds, including 18 new ones, were isolated and structurally determined. The isolated compounds have diverse structures, belonging to 17 chemical classes. Among them, many compounds contain heteroatom, whereas many compounds have complicated configuration due to their flexible structure. The structures and absolute configurations of the new compounds were determined by experimental spectroscopy methods such as HRESIMS, NMR, ECD, single crystal X-ray, along with quantum theoretical calculations for NMR and ECD spectra. 

- The bioactivity test showed that 43 compounds exhibited antimicrobial activity and 14 compounds had cytotoxic activity. Among them, 1 compounds indole-3-carboxylic (OD5) showed remarkable antimicrobial activity against three bacterial strains with MIC values of 16 μg/ml, whereas 6 compounds exhibited noticeable cytotoxic activity with IC50 less than 10 μM. In addition, 2 compounds were also shown to induce apoptosis in human prostate cancer cell DU145 and human lung cancer cell A549. 

- There are scientific records and cultural process for 06 strains of marine microalgae including 04 strains of heterotrophic microalgae Schizochytrium limacinum NT4, Aurantiochytrium mangrovei BT3, Thraustochytrium aureum BT6, Thraustochytrium striatum BT12, and 02 strains of photoautotrophic microalgae Thalassiosira weissflogii KH7 and Nannochloris atomus NT12 to provide biomass for research on secondary metabolites.

- Isolation and structural identification of  71 compounds with 48 different structures from 06 strains of selected marine microalgae, including 19 compounds from Schizochytrium limacinum NT4, 15 compounds from Aurantiochytrium mangrovei BT3, 11 compounds from Thraustochytrium aureum BT6, 12 compounds from Thraustochytrium striatum BT12, 06 compounds from Thalassiosira weissflogii KH7 and 08 compounds from Nannochloris atomus NT12. There are two new compounds including AMHG5 from A. mangrovei BT3 and TWE8 from T. weissflogii KH7. There are 21 isolated compounds showed at least one of antimicrobial, cytotoxic, hypolipidemic and α-glycosidase inhibitory activities. 

- Evaluation of the hypolipidemic effect of the extracts and some isolates from the studied microalgae strains. The results showed that 12 extract residues and 5 compounds exhibited the hypolipidemic effect on the model of HepG2 cell. The mechanism of two potential compounds (24R)-4α-methylstigmasta-7,22-dien-3β-ol (AME1) isolated from A. mangrovei BT3 and cycloartenol (TSH3.1) isolated from T. striatum BT12) with high hypolipidemic effect are determined.

- The antimicrobial activity of the extracts and compounds isolated from selected microalgae were evaluated. The results showed that all the tested extracts and 19 compounds showed activity on at least one tested microorganism strain.

- The cytotoxic activity of extracts and compounds isolated from microalgae were evaluated on four cancer cell lines including A549, HepG2, MCF7 and LNCaP. The results showed that only three extracts (AME, AMH1, TWE) and five compounds (AME8, AMHG7, SLHG7, SLEG7, SLHG2) showed weak and very weak activities.

- The α-glycosidase inhibitory activity of extracts and compounds isolated from T. aureum BT6 were evaluated. The TAH extract and three compounds including TAE15, TAE8 and TAE11 showed activity with IC50 of 48.22±1.94, 26.86±0.29, 7.96±0.75 and 234.4±3.39 µg/mL, respectively.

+ Two compounds IB2 and IB5 not only showed potent cytotoxic activity but also high contents in the sponge Ianthella basta (1.11% and 0.64% w/w in the methanol extract). They could be useful for further applied investigations.

+ Established 02 processes for isolating two compounds with potential cytotoxic and anti-inflammatory activities from a soft coral and a crinoid. Established a process for for culturing a heterotrophic microalga, and a process for producing hydrolyzed seahorse powder.

+ Based on hydrolyzed seahorse powder,successfully prepared and produced the product namely: "Hải mã đan" to enhance health protection.The product has had its circulation declaration dossier prepared according to regulations at theVietnam Food Safety Authority. The product is expected to be approved for circulation after the project finish.

Part 1:

- This is the first report on chemical constituents and their biological activity from 10 sponge species Aaptos aaptos, Amorphinopsis fenestrata, Clathria (Thalysias) reinwardti, Halichondria panicea, Haliclona (Gellius) cymaeformis, Haliclona (Halichoclona) vansoesti, Hippospongia fistulosa (Lendenfeld, 1889), and Rhabdastrella globostellata in Vietnam. Up to date, investigations on chemical constituent of A. fenestrate and H. fistulosa have not been reported worldwide. 

- Isolation and strutural determination of 21 new compounds

- Determination of 5 novel compounds beloging four new carbon skeletons

- Determination absolute configurations of several compounds using experimental circular dichroism (CD) spectroscopy, NMR  analysis and in combination with TDDFT, GIAO theoretical calculation of the spectral data (ECD and NMR) using Spartan18 and Gaussian16 softwares.

Part 2:

+ This is first investigations on chemical constitutents and biological activities of crinoid Comanthus delicata.

+ Isolated and structurally elucidated of 27 new compounds including 10 from crinoid, 03 from sea cucumber, 01 from starfish, and 13 from soft corals.

+ Suggestion that compound CD7 exhibited cytotoxicity through apoptosis caspase 3 induction and compound LOP7 showed  anti-inflammatory activity through inhibition of IL-6, TNF-, COX-2, and iNOS.

Part 3:

+ Discovery of new secondary metabolites from marine bacteria of the South-Central region of Vietnam’s Sea. Accordingly, 5 new secondary metabolites (G666-1, G666-2, G666-3, G631-2 and M893-1) were isolated and characterized.

+ 37 compounds had antimicrobial activity and 1 compound exhibited anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv.

+ The results of this project encourage a research of bioactive compounds from marine microorganisms for their pharmacological uses.

Part 4:

+ Collect samples, study on lipids and biological activities of lipid extracts. build a lipid database for 125 marine organisms collected in the South Central of Vietnam (Khanh Hoa Binh Thuan coastal areas).

+ Successfully researched the process of creating 01 product from black seahorse materials, successfully prepared and produced the product namely: "Hải mã đan" to enhance health protection.

+ Successfully researched the process of preparing phospholipid-based DHA mixture from marine organisms.

Part 5:

+ 18 new compounds were isolated and characterized from 5 samples of marine molluscs of Vietnam.

+ The quantum theoretical calculation methods (DFT NMR and TDDFT ECD) along with experimental spectral methods (HRESIMS, NMR, ECD, X-ray) were used for determination of the complex absolute configurations of many new compounds. 

+ New metabolites tumiduspyrone A (OD12.2) and tumiduspyrone B (OD13) isolated from the sea slug Paromoionchis tumidus were polypropionates possessing rare C17 and C18 carbon skeletons.  

+ The new compound dactylomelanin E (SB03) from the sea slug Aplysia dactylomela was one of the very few bisabolane sesquiterpenes with unusual rearrangement of the side chain. 

+ Compound indole-3-carboxylic (OD5) isolated from the sea slug Paromoionchis tumidus (identical to compound OB39 isolated from the sea snail Maurita arabica) was found to have potential activity against from 3 microorganisms with MIC values of 16 μg/ml. 

+ Six compounds 5α,8α-epidioxy-cholest-6-en-3β-ol (PV6.1), cholest-7-ene-3β,5α,6β-triol (PV7), 11-(3-methylbutanoyl)-3,13-dipropanoyl ilikonapyrone (PV24), 3-acetyl-11-(3-methylbutanoyl)-13-propanoyl ilikonapyrone (PV25), 11,13-dipropanoyl ilikonapyrone (PV32) and the new metabolite verruculatol B (PV31.1) (isolated from the sea slug Peronia verruculata) were found to have good cytotoxic activity against from 1 to 4 cancer cell lines with IC50 value less than 10 μM. Among which, compounds 11-(3-methylbutanoyl)-3,13-dipropanoyl ilikonapyrone (PV24) and 3-acetyl-11-(3-methylbutanoyl)-13-propanoyl ilikonapyrone (PV25) were also shown to induce apoptosis in human prostate cancer cell DU145 and human lung cancer cell A549. 

Part 6:

+ It’s the first time six microalgae species Schizochytrium limacinum, Aurantiochytrium mangrovei, Thraustochytrium aureum, Thraustochytrium striatum, Thalassiosira weissflogii and Nannochloris atomus NT12 have been studied on the secondary metabolites.

+ Two new compounds have been isolated and identified the structure including AMHG5 from A. mangrovei BT3 and TWE8 from T. weissflogii KH7

 Scientific papers in referred journals (list): 29 ISI papers and 19 national publications.

-  Patents: 01 issued patent, 03 invention applications were officially accepted by National Office of Intellectuall Property of Vietnam.

- Technological products (describe in details: technical characteristics, place):

+ Part 1 (stored at Institute of Marine Biochemistry): specimens of the 10 investigated marine sponges; 13 potential bioactive compounds; 84 pure compounds and their NMR data set; reports of isolation and structural elucidation of isolated compounds from the 10 investigated marine sponges; reports of evaluation of isolated compounds’ cytotoxicity on LU-1, MCF-7, HepG2, SK-Mel2, HEK-293A cell lines. Report on determining the content of 06 active compounds in the methanol extracts of selected sponges: IB2 and IB5 from Ianthella basta, CR5 from Clathria (Thalysias) reinwardti; RG1, RG2 and RG8 from Rhabdastrella globostellata;

+ Part 2 (stored at Institute of Marine Biochemistry): specimens of the 30 collected soft corals and echinoderms; 02 potential bioactive compounds; 72 pure compounds and their NMR data set; 07 reports of isolation and structural elucidation of isolated compounds from the 08 researched corals and echinoderms; 01 report of evaluation of cytotoxicity and anti-inflammatory effects of the isolated compounds; 02 process of isolation of potential bioactive compounds CD7 from the crinoid Comanthus delicata and LOP7 from the soft coral Lobophytum pauciflorum. Detailed report on evaluation of cytotoxicity of compound CD7 and anti-inflammatory activity of compound LOP7 regarding mechanism on biological targets.

+ Part 3 (stored at Institute of Marine Biochemistry): 186 pure strains of marine bacteria; 95 pure compounds and their NMR data set; 15 reports of isolation, purification, and fermentation of 15 marine microorganism strains; 15 reports of isolation and structural elucidation of compounds from the 15 investigated microorganism strains; 01 report of evaluation of antimicrobial and anti-tuberculosis effects of the isolated compounds.

+ Part 4 (stored at Institute of Natural Products Chemistry): 21000 capsules of the functional food Hải mã đan; 01 lipid database of 125 marine organisms; 01 process to obtain mixture of DHA phospholipid from marine organism with laborotory scale; 01 process to produce the functional food Hải mã đan.

+ Part 5 (stored at Institute of Marine Biochemistry): specimens of the 28 collected mollusks; 07 potential bioactive compounds; 86 pure compounds and their NMR data set; 07 reports of isolation and structural elucidation of compounds from the 07 investigated mollusks.

+ Part 6 (stored at Institute of Marine Biochemistry): 21 pure compounds and their NMR data set; 06 reports of isolation and structural elucidation of compounds from the 06 researched microalgal strains; 01 report of evaluation of antimicrobial, cytotoxic, and antioxidant effects of the isolated compounds. 01 report on the list marine microalgae species that have been studied to reduce lipid metabolism disorders and results on the effect of reducing lipid metabolism disorders as well as safety levels and initial research on the mechanism of action in reducing lipid metabolism disorders of some compounds isolated from potential microalgae species.

-  Other products: Training: 07 PhD

+ Contituous production of the Hải mã đan functional food for selling in the market.

+ Carry our further studies on fractions and compounds with potential bioactivities directing for further applicative investigations.