INTRODUCTION:

Alzheimer’s disease (AD) is anirreversible neurodegenerative disorder and is suspected to be the most common cause of dementia, mainly affecting older people. The origin of the symptoms is associated with the progressive destruction of cholinergic neurons involved in cognitive function.

One of the strategies to increase the amount of ACh in the nervous system is inhibiting the acetylcholinesterase (AChE) enzyme that rapidly hydrolases the ACh to choline and ethanoic acid. So, cholinesterase (ChE) inhibition is one of the essential therapeutic strategies in Alzheimer's disease treatment.^1–6

Rivastigmine, donepezil,and galanthamine are the most prescribed drug classes for AD treatment as cholinesterase (ChE) inhibitors.⁷ Rivastigmine and galantamine are common examples of naturally derived acetylcholinesterase inhibitors,which have been widely employed as reference compounds in search for more AChE compounds from natural sources. Although most studies agree that donepezil is a safe drug, crucial adverse drug reactions have been reported in the international literature.⁸

Constant exploration, utilizing modern and traditional methods, for natural products that can be translated into more effective and highly selective AD drugs is imperative.⁹ Terrestrial plants have long been used historically to obtain natural remedies.^10,11 Accordingly, the marine environment is a rich and vast underexplored reservoir of bioactive secondary metabolites, with unprecedented mechanisms of action. Marine organisms, such as tunicates, corals, sponges, and algae, and microorganisms, such as marine-derived bacteria, actinomycetes, and fungi, have been promising drug discovery sources.¹²

Various classes of marine-derived compounds have been reported to inhibit key enzymes involved in AD, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), including alkaloids, phenolics, terpenoids, fatty acids, and peptides.¹ The anti-Alzheimer of natural products can only be evaluated following a cholinesterase inhibitory screening of marine animal extracts. For instance, acetylcholinesterase bioactivities of the extracts from 45 species of sponges collected in Mauritius Waters returned two of the most active sponge extracts including sponges Pericharax heterophils and Amphimedon navalis Pulitzer-Finali.¹³ Accordingly, cholinesterase and 5α-reductase inhibitory screening of sponges obtained from Banyuwangi, East Java, Indonesia, resulted in three extracts that showed 61.21% inhibition, including Callyspongia sp., Niphatesolema, and Agelas Nakamura.¹⁴ In the light of those findings, we aimed to screen ChE inhibitory from marine sponges collected from Balinese waters, under-investigated waters but rich in species biodiversity.

MATERIALS AND METHODS:

Collection of sponges:

The sponges were collected by hand using SCUBA Diving at depths between 5-20 m from the coast of Tulamben, Bali, Indonesia, in December 2020. The specimens were identified by Dr.rer.nat Edwin Setiawan and the vouchers are available for inspection at the Department of Biology, ITS, Indonesia. The freshly collected sponges were kept frozen at -26 °C until the following experiment. The frozen animal (50 g, dry weight) for each sponge was thawed, diced, and extracted with a mixture of methanol and dichloromethane (1:1, 500 mL). The mixture was filtered, and the residue was extracted twice with the same volume of solvent mixture. The combined filtrates were concentrated to return crude extracts.

Cholinesterase inhibitory assay:

The cholinesterase inhibitory test was performed based on the modified Ellman’s method.¹⁵ Initial methanolic sponge extracts (1 mg/mL) were prepared and diluted in microplate well to a final 100µg/mL test concentration. Next, 1.5mM ATCI (25 µL), 3 mM DTNB (125 µL),Tris buffer (50 µL), and 25 µL of 0.22 U/mL of EeAChE enzyme were added to the microplate and shaken for 30 s in a microplate reader (Bio-Tek Instrument, USA). Accordingly, galantamine was prepared as a positive control, and 10 % methanol was used as a blank, the experiment wascompleted in triplicates. Finally, the percentage inhibitory was determined by using the following formula:

% Inhibition = [(Mean velocity of controlMean velocity of sample)/Mean velocity of control] × 100. The IC₅₀values werecalculated by GraphPad Prism 8.0 software by plotting the log concentration of samples as axis and % inhibition as ordinate.

Isolation and identification of metabolites:

The organic crude extract (⁓1.3 g) was submitted to liquid-liquid extraction by partitioning between n-hexane and water and by repartitioning the water layer with ethyl acetate (EtOAc) to fractionate the extract in an n-hexane (300 mg), EtOAc (600 mg), and aqueous (400 mg, mainly salts) soluble portions. Then, an aliquot of the EtOAc fraction (200 mg) was subjected to Sephadex LH-20 chromatography by using 100% methanol as eluent to yield three main fractions (Fr 12-14) that were combined (60 mg) and further purified by solid phase extraction (SPE) by loading on a C18 cartridge (M-N Chromabond C18 6 mL/500 mg). The fraction eluted with 10% of MeOH in water contained pure helenaquinolsulfate (50 mg) by NMR analysis.

RESULTS AND DISCUSSION:

Cholinesterase inhibitory activity:

The crude extracts of ten sponges displayed AchE inhibition ranging from 24.6 to 95.17 % (Table 1). At a concentration of 100 μg/mL, a sponge extract of Petrosia nigricans (Lindgren, 1897) displayed the strongest inhibition at 95.17 % and the weakest shown by the sponge Callyspongia (Callyspongia) sp. Duchassaing & Michelotti, 1864 at 24.60 %. Another Petrosia sponge (SPT 14) also displayed a quite significant inhibition during the screening, but it was a much smaller value than that of the SPT 10.

Table 1: The inhibitory activities of the organic extracts of marine sponges against acetylcholinesterase (AChE)

Code	Sponge	%AChE Inhibition
SPT 1	Petrosia (Petrosia) sp 1. Vosmaer, 1885	40.22±10.43
SPT 3	Paratetilla bacca (Selenka, 1867)	40.46±1.27
SPT 10	Petrosia nigricans (Lindgren, 1897)	95.17±0.64
SPT 12	Callyspongia (Callyspongia) sp. Duchassaing& Michelotti, 1864	24.60±0.64
SPT 13	Haliclona (Haliclona) sp. Grant, 1836	32.63±2.19
SPT 14	Petrosia (Petrosia) sp 2. Vosmaer, 1885	67.37±2.19
SPT 15	Callyspongia (Toxochalina) sp. Ridley, 1884	35.83±1.18
SPT 16	Xestospongia testudinaria (Lamarck, 1815)	35.44±2.25
SPT 17	Spheciospongiainconstans (Dendy, 1887)	37.15±2.72
SPT 20	Haliclona(Flagellia) sp. Van Soest, 2017	29.09±0.06

Data presented as mean ± SD of three independent experiments, each done in triplicate. Samples were tested at 100 μg/mL.

Guided by the strongest inhibition value of the Petrosia nigricans, further liquid-liquid fractionations were performed. Theresulting hexane and ethyl acetate fractions showed AChE IC₅₀ values of 2.01±0.04 and 0.14±0.003µg/mL, respectively (Table 2). The Ethyl acetate fraction displayed much stronger activity compared to that of the crude extract (1.41±0.02µg/mL) and the standard galantamine (0.45±0.13) (Figure 1). This indicates that the semipolar bioactive secondary metabolites may be responsible for the AChEshown by the original crude extract.

Table 2: IC₅₀ values of Petrosia nigricans extract and fractions against acetylcholinesterase

Sample	IC₅₀ (µg/mL)
Extract	1.41 ± 0.02
n-Hexane fraction	2.01 ± 0.04
Ethyl acetate Fraction	0.14 ± 0.003
Galantamine	0.45 ± 0.13

Figure 1: Dose-dependent response of selected marine sponges to the inhibition of acetylcholinesterase. Data presented as mean± standard deviation of three independent experiments.

Subsequent chromatographic purification of the EtOAc fraction resulted in the identification of a known pentacyclic hydroquinone, halenaquinolsulfate. The compound exhibited AChE IC₅₀ values of 40.73±0.19 µg/mL, much bigger than that of the EtOAc fraction from which the HQS was purified (Figure 2a). The drastic decline of the AChE inhibition from the fraction to the purified compound may be associated with the synergetic effect exerted by several vital compounds in the fractions.¹⁶

BuChE, another enzyme belonging to the group of cholinesterase group, was also considered to act as a compensatory mechanism for acetylcholine metabolism.^17,18Therefore, the purified compound HQS was submitted for BuChE inhibitory test. The assay indicated that the compound inhibited BuChEwith an IC₅₀ value equal to 53.12±0.32µg/mL, slightly more significant than that of the AChE value (Figure 2b).

(a) AChE 40.73±0.19µg/Ml

(b) BuChE 53.12±0.32µg/mL

Figure 2: Dose-dependent response of halenaquinolsulfate against AChE (a) and BuChE (b)

An initial screening of sponge extracts against cholinesterase and 5α-reductase enzymes had been completed by Suciati et al.The sponge specimens were harvested fromthe tropical Tabuhan Island, East Java, Indonesia.¹⁴ The organic extracts of the three species namely, Callyspongia sp., Niphatesolema, and Agelas Nakamura showed significant inhibition against the AChE enzymes. Among them, A. Nakamuraexhibited the highest inhibition with an IC₅₀ value of 1.05μg/mL. Interestingly, a methanolic extract of Petrosia sp. displayed 61.21% inhibition against 5α-reductase; however, no further chemical study was performed on the extract.

Isolation and purification of secondary metabolite:

Subsequent chromatographic separation of the EtOAc fraction of the P. nigricans returned a known pentacyclic hydroquinone, HQS. The structure of this compound was concluded by the comparison of the ¹H, ¹³C NMR chemical shifts, and MS data with those reported in the literature.¹⁹ Five distinctive downfield protons are clearly visible from the proton spectrum of HQS, together with a characteristic methyl singlet at δ_H 1.64 ppm (CH₃-20). The two coupled doublet at δ_H 7.46 (H-15) and δ_H 6.77 (H-14) were assigned to the two aromatic protons adjacent to the characteristic quaternary carbons bearing the sulfate group. The most downfield signal δ_H 9.09 ppm was identified as the isolated aromatic proton (H-11), while the distinctive furan proton (H-1) appeared as a singlet at δ_H 8.43 ppm (Figure 3). The MS spectrum of HQSdisplayed an ion adduct at m/z of 459 [M+Na]⁺ that is in full agreement with that in the literature.

Figure 3: ¹H NMR of HQS (MeOD, 500 MHz)

Halenaquinolsulfate was first isolated from the Okinawan marine sponge Xestospongia sapra.¹⁹ Subsequently, the absolute stereostructure of HQS was determined as (12bS)-(+) halenaquinolsulfateby theoretical calculation of CD spectra.²⁰ Next, the natural product was also identified from an extract of the Indo-Pacific sponge Xestospongia cf. carbonaria.²¹ It was later co-eluted as a minor component of the Xestospongia sp. obtained from Fiji Islands.²² The hydroquinone displayed a micromolar affinity for human Tyrosyl-DNA Phosphodiesterase (TDP1) inhibitor²³, inhibited the activity of eukaryotic DNA polymerases in varying degrees,²⁴ and prevented cell membrane fusion events of echinoderm gametes.²⁵

Halenaquinolsulfatehas never been reported from the sponge Petrosia nigricans.²⁶ Instead, P. nigricans were reported to produce purine analogs (nigricines)²⁷ and a biscembranoid, petronigrione²⁸. Recently, the extract of P. nigricansharvested from The Philippines returned two new sarasinosides, 5,8-epoxysarasinoside, and 8,9-epoxysarasinoside, that displayed low cytotoxicity against the HCT116 (colon) and A549 (lung) cancer cell lines.²⁹

Although HQS has never been isolated from the sponge Petrosia nigricans, the genus Petrosia has produced a series of pentacyclic quinone with furan moiety, including menaquinone. For instance, A series of menaquinone derivatives, including eight monomers, six dimers, and two trimers, were isolated from P. alfiani collected in Indonesia.³⁰ Interestingly, all this monoterpenoids class of compounds isolated from Petrosia sponges is from sponges collected in tropical areas. However, pentacyclic hydroquinone is not exclusively the metabolite of Petrolia. For example, pentacyclic hydroquinone, halenaquinol, was identified from both genera, including Petrosia (P. seriata)³¹ and Xestospongia (X. exigua and X. supra).²⁰ One simple reason for this sharing of metabolites between two genera wasthe biosynthetic origin of the menaquinone family, which appears to be a mixture of sesquiterpene and quinone instead of polyketide origin.²¹

Next, HQS has never been reported to inhibit key enzymes involved in AD, including AChE, BuChE, β-secretase (BACE-1), and different kinases.Therefore, this finding would expand the bioactivity of pentacyclic hydroquinone.

CONCLUSION:

Guided by cholinesterase inhibitory assay, a known pentacyclic hydroquinone, halenaquinolsulfate, was successfully isolated from an extract of tropical sponge Petrosia nigricans. The metabolite displayed a moderate acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

We thank Universitas Pendidikan Ganesha, Indonesia, for financially supporting I.W.M. under PKLN scheme No. 1168/UN48.16/LT/2023.

REFERENCES:

1. Ghoran SH, Kijjoa A. Marine-Derived Compounds with Anti-Alzheimer’s Disease Activities. Mar Drugs. 2021; 19(8): 410. https://doi.org/10.3390/md19080410.

2. Jadhav RP, Kengar MD, Narule O V., Koli VW, Kumbhar SB. A Review on Alzheimer’s Disease (AD) and its Herbal Treatment of Alzheimer’s Disease. Asian J Res Pharm Sci. 2019; 9(2): 112. https://doi.org/10.5958/2231-5659.2019.00017.1.

3. Kumar DR, Shankar MS, Reddy PP, Kumar BRS, Sumalatha N. A Review on Alzheimer’s Disease. Res. J. Pharmacol Pharmacodynamics. 2014; 6(1): 59–63.

4. Dhinakaran S, Tamilanban T, Chitra V. Targets for Alzheimer’s Disease. Res. J. Pharm. Technol. 2019; 12(6): 3073. https://doi.org/10.5958/0974-360X.2019.00521.3.

5. Dhananjayan K, Sumathy A, Palanisamy S. Molecular Docking Studies and in-vitro Acetylcholinesterase Inhibition by Terpenoids and Flavonoids. Asian. J. Res. Chem. 2013; 6(11): 1011–7.

6. Sandeep Reddy CH, Sree Kumar Reddy G, Mahto MK, Kunala P, Chaitanya Kanth R. Insilico design and discovery of some novel ache inhibitors for treatment of Alzheimer’s disorder. Res. J. Pharm. Technol. 2012; 5(3): 424–7.

7. Cummings JL, Tong G, Ballard C. Treatment Combinations for Alzheimer’s Disease: Current and Future Pharmacotherapy Options. J Alzheimer’s Dis. 2019; 67(3): 779–94. https://doi.org/10.3233/JAD-180766.

8. Cacabelos R. Donepezil in Alzheimer’s disease: From conventional trials to pharmacogenetics. Neuropsychiatr Dis Treat 2007; 3(3): 303–33.

9. Haake A, Nguyen K, Friedman L, Chakkamparambil B, Grossberg GT. An update on the utility and safety of cholinesterase inhibitors for the treatment of Alzheimer’s disease. Expert Opin Drug Saf. 2020; 19(2): 147–57. https://doi.org/10.1080/14740338.2020.1721456.

10. Athitya LVS, Bharath VMV, Nellore J, Prakash P. Screening of Gracilaria corticata Extracts for Acetylcholinesterase Inhibitory Activity. Res. J. Pharm. Technol. 2018; 11(9): 3848. https://doi.org/10.5958/0974-360X.2018.00704.7.

11. Chitra V, Narayanan J. In vitro Screening for Anti-Cholinesterase and Anti Oxidant Activity of Extract of Garcinia hanburyi. Res. J. Pharm. Technol. 2018; 11(7): 2918. https://doi.org/10.5958/0974-360X.2018.00538.3.

12. Fristiohady A, Malaka MH, Safitri ARW, et al. Anti-inflammatory activity of ethanol extract of marine sponge petrosia sp. By supression the level of tumor necrosis factor-alpha. Res J Pharm Technol 2021; 14(8): 4435–9. https://doi.org/10.52711/0974-360X.2021.00770.

13. Beedessee G, Ramanjooloo A, Surnam-Boodhun R, van Soest RWM, Marie DEP. Acetylcholinesterase-Inhibitory Activities of the Extracts from Sponges Collected in Mauritius Waters. Chem Biodivers 2013; 10(3): 442–51. https://doi.org/10.1002/cbdv.201200343.

14. Suciati, Rabgay K, Fachrunniza Y, et al. Enzyme inhibitory activities of marine sponges against cholineesterase and 5α-reductase. Malays Appl Biol. 2019; 48(3): 77–8.

15. Komersová A, Komers K, Čegan A. New Findings about Ellman’s Method to Determine Cholinesterase Activity. Zeitschrift Für Naturforsch C 2007;62(1–2):150–4. https://doi.org/10.1515/znc-2007-1-225.

16. Bhanukiran K, Singh R, T A G, et al. Vasicinone, a pyrroloquinazoline alkaloid from Adhatoda vasica Nees enhances memory and cognition by inhibiting cholinesterases in Alzheimer’s disease. Phytomedicine Plus 2023; 3(2): 100439. https://doi.org/10.1016/j.phyplu.2023.100439.

17. Andrisano V, Naldi M, De Simone A, Bartolini M. A patent review of butyrylcholinesterase inhibitors and reactivators 2010–2017. Expert Opin Ther Pat 2018; 28(6): 455–65. https://doi.org/10.1080/13543776.2018.1476494.

18. Košak U, Brus B, Knez D, et al. The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity. J Med Chem 2018; 61(1): 119–39. https://doi.org/10.1021/acs.jmedchem.7b01086.

19. Kobayashi M, Shimizu N, Kyogoku Y, Kitagawa I. Halenaquinol and halenaquinol sulfate, pentacyclic hydroquinones from the Okinawan marine sponge Xestospongia sapra. Chem Pharm Bull 1985; 33(3): 1305–8. https://doi.org/10.1248/cpb.33.1305.

20. Harada N, Uda H, Kobayashi M, Shimizu N, Kitagawa I. Absolute stereochemistry of the halenaquinol family, marine natural products with a novel pentacyclic skeleton, as determined by the theoretical calculation of circular dichroism spectra. J Am Chem Soc. 1989; 111(15): 5668–74. https://doi.org/10.1021/ja00197a025.

21. Alvi KA, Rodriguez J, Diaz MC, et al. Protein tyrosine kinase inhibitory properties of planar polycyclics obtained from the marine sponge Xestospongia cf. carbonaria and from total synthesis. J. Org. Chem. 1993; 58(18): 4871–80. https://doi.org/10.1021/jo00070a023.

22. Longeon A, Copp BR, Roué M, et al. New bioactive halenaquinone derivatives from South Pacific marine sponges of the genus Xestospongia. Bioorg Med Chem 2010; 18(16): 6006–11. https://doi.org/10.1016/j.bmc.2010.06.066.

23. Bermingham A, Price E, Marchand C, et al. Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase (TDP1). SLAS Discov 2017; 22(9): 1093–105. https://doi.org/10.1177/2472555217717200.

24. Shioda M, Kano K, Kobayashi M, et al. Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p -hydroquinone sulfate obtained from a marine sponge. FEBS Lett. 1994; 350(2–3): 249–52. https://doi.org/10.1016/0014-5793(94)00777-2.

25. Ikegami S, Kajiyama N, Ozaki Y, et al. Selective inhibition of membrane fusion events in echinoderm gametes and embryos by halenaquinol sulfate. FEBS Lett 1992; 302(3): 284–6. https://doi.org/10.1016/0014-5793(92)80460-X.

26. Lee Y-J, Cho Y, Tran HNK. Secondary Metabolites from the Marine Sponges of the Genus Petrosia: A Literature Review of 43 Years of Research. Mar Drugs 2021; 19(3): 122. https://doi.org/10.3390/md19030122.

27. Ashour M, Edrada-Ebel R, Ebel R, Wray V, van Soest RWM, Proksch P. New Purine Derivatives from the Marine Sponge Petrosia nigricans. Nat Prod Commun. 2008; 3(11): 1934578X0800301. https://doi.org/10.1177/1934578X0800301119.

28. Nhiem NX, Van Quang N, Van Minh C, et al. Biscembranoids from the Marine Sponge Petrosia Nigricans. Nat Prod Commun 2013; 8(9): 1934578X1300800. https://doi.org/10.1177/1934578X1300800905.

29. Mama RL, Gelani CD, Daluz JMT, Uy MM, Ohta E, Ohta S. Two new sarasinosides from marine sponge Petrosia nigricans. Nat Prod Res. 2023: 1–9. https://doi.org/10.1080/14786419.2023.2175359.

30. Tanokashira N, Kukita S, Kato H, et al. Petroquinones: trimeric and dimeric xestoquinone derivatives isolated from the marine sponge Petrosia alfiani. Tetrahedron. 2016; 72(35): 5530–40. https://doi.org/10.1016/j.tet.2016.07.045.

31. Gorshkova IA, Gorshkov BA, Fedoreev SA, Shestak OP, Novikov VL, Stonik VA. Inhibition of membrane transport ATPases by halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata. Comp Biochem Physiol Part C Pharmacol Toxicol Endocrinol. 1999; 122(1): 93–9. https://doi.org/10.1016/S0742-8413(98)10084-1.

Received on 31.08.2023 Modified on 18.11.2023

Research J. Pharm. and Tech 2024; 17(7):3329-3333.

DOI: 10.52711/0974-360X.2024.00520