Study on exploitation of sponge medicinal materials at the sea areas of Centeral Vietnam (the sea areas from north to middle Central Vietnam) targeting on cytotoxic activity, using for treatment/assisted treatment of cancer

Project leader's name
Assoc.Prof. Phan Van Kiem
Research hosting institution
Institute of Marine Biochemistry
Project duration
2016 - 2018
́Project’s budget
7.250 millions VNĐ
Classify
Excellent
Goal and objectives of the project

- Collection of 10 sponge samples at the sea areas from north to middle Central Vietnam and identifiation their scientific name.
- Extraction, isolation, and structural elucidation of chemical constituents from 10 sponge species.
- Evaluation in vitro cytotoxic activity of the isolated compounds, study on cytotoxic mechanism of the lead compounds via apoptosis and cell cycle arrest pathways.
- Establishment of a procedure in the preparation of fraction PĐ1 (rich in active compounds) from selected sponge species.
- Determination of acute, subacute toxicities, and evaluation in vivo antitumor activity of the fraction PĐ1

Main results

- Scientific results:
+ The 10 sponge species including Acanthella klethra (Pulitzer-Finali, 1982), Agelas oroides (Schmidt, 1864), Amphimedon complanata (Duchassaing, 1850), Haliclona xena (De Weerdt, 1986), Hippospongia pacifica (Hyatt, 1877), Ircinia ramosa (Keller, 1889), Polymastia boletiformis (Lamarck, 1815), Rhabdastrella providentiae (Dendy, 1916), Smenospongia cerebriformis (Duchassaing & Michelotti, 1864), và Xestospongia muta (Schmidt, 1870) were collected and archived their voucher specimens. Some of them such as Agelas oroides, Xestospongia muta, Haliclona xena, Polymastia boletiformis, Smenospongia cerebriformis, Ircinia ramose were taxonomical identification by analysis of their DNA indicator and comparison with global gen bank.
+ From 10 sponge species, 94 substances were isolated using chromatographic methods. Their structure were identify to be 87 different compounds by moder spectrosopic methods such as HR-ESI-MS, 1D- and 2D-NMR, Circular dichroism spectroscopy. Among 87 obtained compounds, there were 21 new compounds and 8 other compounds have not been reported on the stereo chemistry. 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 macrocyclic bis quinolizidine alkaloid and indole alkaloid. Some compounds belonging phenolic and indole alkaloid were found in several sponge species.
In partitucally, 24 compounds (8 new compounds and 4 stereogenic structures) isolated from Smenospongia cerebriformis, 4 compounds isolated from Haliclona xena, 5 compounds isolated from Hippospongia pacifica (1 stereogenic structure), 21 compounds (12 new compounds including 1 novel compound) isolated from Rhabdastrella providentiae, 5 compounds isolated from Amphimedon complanata, 4 compounds isolated from Polymastia boletifomis, 6 compounds isolated from Ircinia ramosa, 7 compounds isolated from Acanthella klethra, 7 compounds isolated from Agelas oroides, 11 compounds (1 new compound and 3 stereogenic structures) isolated from Xestospongia muta.
+ The isolated compounds were evaluated their cytotoxic activity towards LU-1, MCF-7, HepG2, SK-Mel2, HL-60, and NIH/3T3 cell lines. Nine compounds inculing SC8, SC16, RP2, XM1-XM6 exhibited potent cytotoxic activity with their IC50 lower than 20 µM. Especially, compounds XM1 and XM2 showed IC50 values ( 1 µM) ower than positive control ellipticine.
+ Compounds SC8, SC16, XM1, XM2 were selected for study cytotoxic mechanism and showed that their cytotoxic activities were partly explained by induction of apoptosis in LU-1 cells. Compound XM1 was also selected for evaluation on the cell cycle arrest and effect on protein markers assited apoptosis. The results indicated that compound XM1 arrested phase G2 in the cell cycle, enhenced caspase 3 and Bax, but suppressed activation of Bcl-2.
+ Compounds SC8, SC16, XM1, XM2 were evaluated their effect on TNF-α secretion in HepG2 cells. Compound SC8 inhibited TNF-α secretion at concentration of 18 µg/mL. Compounds SC16 inhibited TNF-α secretion at all tested concentrations of 2, 4, and 8 µg/mL. Compounds XM1 and XM2 inhibited TNF-α secretion at diluted concentrations of 0.75 and 0.5 µM but they showed enhenced TNF-α secretion at concentration as high as 1.0 µM.
+ Compounds SC8, SC13, SC16 remarkably inhibited NO production in activated RAW264.7 cells with IC50 values of 16.66±3.54, 10.40±1.28 and 16.66±3.54 µM. Compounds RP2, RP7, RP9-RP12, RP14, and RP-15 inhibited NO production in activated BV2 cells with IC50 values in range from 7.4 ± 0.4 to 39.2 ± 2.0 μM.
+. Fraction PĐ1 was found safe in actue and subacute toxic evaluation on mouse model. A LD50 value was not determined upto experimental dose of 6000 mg/kgP/day. All of tested animal were saved in case of oral administration of PĐ1 upto a dose of 600 mg/kgP/day during 30 days
- Applied results:
+ A procedure for preparation of fraction PĐ1 from Xestospongia muta (3 kg dried sample, 100 g MeOH extract) was established and applied to yield 88.5 g product (Yield = 0.295%, w/w dried sample) for in vivo study

Some images of the project:

Anh dai dien VAST TĐ DLB 01 16 18

Novelty and actuality and scientific meaningfulness of the results

- This is the first report on chemical constituents and their biological activity from 10 sponge species A. klethra, A. oroides, A. complanata, H. xena, H. pacifica, I. ramosa, P. boletiformis, R. providentiae, S. cerebriformis, and X. muta in Vietnam. Up to date, investigations on chemical constituent of H. xena, H. pacifica, and R. providentiae have not been report worldwide.
- Isolation and strutural determination of 21 new compounds
- Determination of strereo chemistry of additional 8 compounds which have not been reported to date.
- Purification of two geometric isomers (globostelletins C and D) which were prevously reported in a mixture.
- Determination absolute structure of several compounds using circular dichroism (CD) spectroscopy, the first CD spectrometer in Vietnam.

Products of the project

- Scientific papers in referred journals (list):
a) ISI papers
[1]. Phan Van Kiem, Le Thi Huyen, Dan Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Pham Van Cuong, Tran Hong Quang, Chau Van Minh, Nguyen Van Dau, Young Ah Kim, Lalita Subedi, Sun Yeou Kim, Seung Hyun Kim, Sesquiterpene derivatives from marine sponge Smenospongia cerebriformis and their anti-inflammatory activity, Bioorganic & Medicinal Chemistry Letters, 27, 1525–1529 (2017)
[2]. Le Thi Huyen, Dan Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Tran Hong Quang, Pham Hai Yen, Chau Van Minh, Nguyen Van Dau and Phan Van Kiem, Sesquiterpene quinones and diterpenes from Smenospongia cerebriformis and their cytotoxic activity, Natural Product Communications, 12, 477-478 (2017)
[3]. Le Thi Huyen, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Pham Hai Yen, Hoang Le Tuan Anh, Tran Hong Quang, Bui Huu Tai, Nguyen Van Dau, and Phan Van Kiem, Naphtoquinones and sesquiterpene cyclopentenones from the sponge Smenospongia cerebriformis with their cytotoxic activity. Chemical & Pharmaceutical Bulletin, 65, 589-592 (2017)
[4]. Duong Thi Dung, Pham Hai Yen, Nguyen Xuan Nhiem, Tran Hong Quang, Bui Huu Tai, Chau Van Minh, Dong Cheol Kim, Hyuncheol Oh, Youn Chul Kim and Phan Van Kiem, New acetylated terpenoids from sponge Rhabdastrella providentiae inhibit NO production in LPS stimulated BV2 cells, Natural Product Communications, 13, 661-664 (2018)
[5]. Phan Van Kiem, Duong Thi Dung, Pham Hai Yen, Nguyen Xuan Nhiem, Tran Hong Quang, Bui Huu Tai, Chau Van Minh, New isomalabaricane analogues from the sponge Rhabdastrella providentiae and their cytotoxic activities, Phytochemistry Letters, 26, 199–204 (2018)
[6]. Duong Thi Dung, Dan Thi Thuy Hang, Pham Hai Yen, Tran Hong Quang, Nguyen Xuan Nhiem, Bui Huu Tai, Chau Van Minh, Youn-Chul Kim, Dong Cheol Kim, Hyuncheol Oh, and Phan Van Kiem, Macrocyclic bis-quinolizidine alkaloids from Xestospongia muta, Natural Product Research, In press. https://doi.org/10.1080/14786419.2018.1455043.
[7]. Duong Thi Dung, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Tran Hong Quang, Bui Huu Tai, Pham Hai Yen, Nguyen Thi Hoai, Do Cong Thung, Chau Van Minh and Phan Van Kiem, Rhabdaprovidines D–G, Four new 6,6,5-tricyclic terpenoids from the Vietnamese sponge Rhabdastrella providentiae, Natural Product Communications, 13(10), 1251-1254 (2018).
[8]. Phan Van Kiem, Duong Thi Dung, Do Thi Trang, Tran Hong Quang, Nguyen Thi Thanh Ngan, Tran Minh Ha, Hoang Le Tuan Anh, Pham Hai Yen, Do Thi Thao, Nguyen Xuan Nhiem, Bui Huu Tai, Hyuncheol Oh, Youn Chul Kim, and Chau Van Minh. Constituents from Ircinia echinata and their Antiproliferative Effect on Six Human Cancer Cell Strains, Letters in Organic Chemistry, 14, 248-253 (2017).
b) Domestic scintific papers
[1]. Le Thi Huyen, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Pham Hai Yen, Nguyen Van Dau, Chau Van Minh, Phan Van Kiem, Sesquiterpene phenols from marine sponge Smenospongia cerebriformis, Vietnam Journal of Chemistry, 55(2) 148-152 (2017)
[2]. Le Thi Huyen, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Pham Hai Yen, Nguyen Van Dau, Chau Van Minh, Phan Van Kiem, Merosesquiterpenes from marine sponge Smenospongia cerebriformis, Vietnam Journal of Chemistry, 55(2) 153-157 (2017)
[3]. Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai, Pham Hai Yen, Hoang Le Tuan Anh, Do Thi Trang, Duong Thi Hai Yen, Tran Hong Quang, Chau Van Minh, Phan Van Kiem, Terpenoid metabolites from the sponge Ircinia ramosa, Vietnam Journal of Chemistry, 55(6e), 2017, 1-6 (2017)
[4]. Duong Thi Dung, Nguyen Xuan Nhiem, Do Thi Trang, Pham Hai Yen, Tran Hong Quang, Hoang Le Tuan Anh, Do Cong Thung, Bui Huu Tai, Chau Van Minh, Phan Van Kiem, Isolation of isomalabaricane analog from the sponge Rhabdastrella providentiae, Vietnam Journal of Chemistry, 55(6e), 2017, 38-41 (2017)
[5]. Duong Thi Dung, Nguyen Thi Cuc, Pham Hai Yen, Nguyen Xuan Nhiem, Tran Hong Quang, Hoang Le Tuan Anh, Do Cong Thung, Do Thi Thao, Bui Huu Tai, Chau Van Minh, Phan Van Kiem, Petrosin and petrosin A from the Vietnamese sponge Xestospongia muta, Vietnam Journal of Chemistry, 55(6e), 2017, 72-75 (2017)
[6]. Le Thi Huyen, Dan Thi Thuy Hang, Duong Thi Hai Yen, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Pham Hai Yen, Nguyen Van Dau, Chau Van Minh, Phan Van Kiem, Two dolastane diterpenoids from marine sponge Smenospongia cerebriformis, Vietnam Journal of Chemistry, 55(6e), 2017, 94-97 (2017)
[7]. Dương Thị Dung, Đan Thị Thúy Hằng, Phạm Hải Yến, Nguyễn Xuân Nhiệm, Trần Hồng Quang, Bùi Hữu Tài, Phan Văn Kiệm, Nghiên cứu thành phần hóa học loài hải miên Rhabdastrella providentiae, Tạp Chí Hóa học, 56(1), 81-85 (2018)
[8]. Đan Thị Thúy Hằng, Dương Thị Dung, Trần Hồng Quang, Phạm Hải Yến, Nguyễn Xuân Nhiệm, Bùi Hữu Tài, Đỗ Công Thung, Châu Văn Minh, Phan Văn Kiệm. Một số hợp chất alkaloid từ loài hải miên Agelas oroides. Tạp Chí Hóa học, 56(3), 368-372 (2018)
- Patents (list):
[1] Patent name: Isomalabaricane-type compounds and their extraction methods from the sponge Rhabdastrella providentiae, Application number 1-2018-00541, Accepted application by the Decision No. 19094/QĐ-SHTT in March 26, 2018.
- Technological products (Products are deposited at the Institute of Marine Biochemistry):
[1]. 10 voucher speciments of sponge species.
[2]. Reports on the isolation of compounds from 10 sponge species
[3]. Reports on the structural determination of the isolated compounds
[4]. Reports on the biological activity of isolated compounds including cytotoxic activity; cytotoxic mechanism by apoptosis and cell cycle arrest pathways; effect on TNF- secretion; inhibition of NO production in the LPS-stimulated BV2 and RAW264.7 cells; results of acute, subacute toxicities, and in vivo antitumor activity of the fraction PĐ1
[5]. Procedure for preparation of the fraction PĐ1 from the sponge Xestospongia muta
- Other products (if applicable): Graduate education
[1]. Dr. Le Thi Huyen, Graduated PhD student, Hanoi University of Science.
[2]. Duong Thi Dung, 3rd year PhD student, Graduate University of Science and Technology

Place of application

Our results recommend for deverlopment of anti-cancer products from the sponge Xestospongia muta which was clarified on chemical constituents, cytotoxic activity, cytotoxic mechanism, acute and subacute toxic evaluation, and in vivo study.

Recommendations

-The Vietnamese sponges should receive more attention by researchers to find new compound, new lead compound for new drug discovery and deverlopment.
- Nine promising cytotoxic compounds in this study should be required further evaluation to confirm and clarify their cytotoxic machanism. These compounds should be submited to the library of lead compounds for anticancer agents.
- In vivo study should be done for compound XM1, the most active compound and could be isolated in gram scale from Xestospongia muta.
- Sevaral compounds from the sponges Smenospongia cerebriformis and Rhabdastrella providentia inhibited the production of NO in BV2 or RAW264.7 cells. They could be useful for treatment of inflamation, thus, further study should be required.

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