INTRODUCTION:

The search for novelty had led mankind to explore the unlikeliest crevices of our planet. Novelty is what fuels our minds to think the unthinkable and imagine the unimaginable. Many of the native herbs have helped the humanity in combating the ailments from times immemorial. Just to name a few varied categories, we can consider the examples of different varieties of bitter gourd, neem and black plums^1,2,3. From understanding the surrounding habitat to developing a systematic and scientific method of treatment has been the contribution of our ancient scientists (Rishis) – in the name of Ayurveda^4,5.

One of the aspects of this habitat is water. Mother nature’s treasures are enormous and the biggest of her jewels is the mighty ocean, one of the most diverse biomasses in the world, covering an astounding 71% of the earth's surface and representing over 95% of the biosphere⁶. This expanse of marine life presents a fertile area for opportunities in research. Although largely untapped, marine natural products have been recognized as a powerful bioweapon to fill the pipeline with drug leads for the pharmaceutical industry^7,8,9and one which we have explored in the course of this study. Cnidaria represents a phylum of marine organisms consisting of colony-forming aquatic animals which have a rich potential to be a source of drugs and therapeutic agents^10,11. Our study placed importance on the tiny but tough, flower-like animals, called the Zoanthids (Fig. No.1), that are found carpeting rocky and rubble areas, adapted to be regularly exposed to the air during low tide.

Fig. No.1 Mat of zoanthids camouflaged on the rocks

There are multiple reasons why zoanthids, belonging to the order Zoantharia, standout among other aquatic life-forms¹². Firstly, their unusual lifestyle, symbiotic photosynthesis and, the ability to form colonies with polyps as well as the marine niche which they occupy make them an interesting organism and awakens the curiosity of research scientists^12,13. Moreover, the presence of certain biochemical substances which are of pharmaceutical significance has sparked interest as a potential therapeutic agent. Some important bioactive compounds isolated from zoanthids include zoanthoxanthins, zoanthoamines^14,15, a group of alkaloids as well as one of the deadliest marine toxins called Palytoxin, isolated from zoanthid Palythoa species^15,16.

The published scientific reports on zoanthids from the Indian coast are few and deal mainly with its identification. Although one such study carried out in the year 2013¹⁷, reported for the first time the identification of Zoanthus sansibaricus (accession number HQ840729) through molecular and morphological data, there is limited scientific data reported on the isolation of chemical constituents from the Zoanthus sansibaricus of the Indian coast.

Scientific classification of Zoanthus sansibaricus¹⁸

Kingdom	:	Animalia
Phylum	:	Cnidaria
Class	:	Anthozoa
Subclass	:	Hexacorallia
Order	:	Zoantharia
Suborder	:	Brachycnemina
Family	:	Zoanthidae
Genus	:	Zoanthus
Species	:	sansibaricus

Studying the phytochemical profile of a crude drug is of prime concern before its full potential as a substance of therapeutic use can be completely correlated^19,20. Understanding the phytochemical profile and characterization inturn can be successfully performed only after an elaborate and planned techniques of isolation of these bioconstituents from the biosource^21,22,23.

The aim of the study is to first isolate chemical constituents from the Zoanthus sansibaricus, followed by characterization of isolated constituents by UV, IR absorption studies, ¹H NMR and ¹³C NMR and Mass analysis. Furthermore, the antimicrobial screening of Zoanthus fractions and estimation of total phenolic and total flavonoid content has also been performed.

MATERIALS AND METHODS:

Preparation of extract:

Dried methanolic extract (A-001) and dried methanol: chloroform (1:1) (A-002) extract of Zoanthus sp. was obtained from the National Institute of Oceanography (NIO), Goa.

Sample details:

1) NIO-971(A-001), Zoanthus species LD-3, 10/05/2010

2) NIO-971(A-002), Zoanthus species LD-3, 13/05/2010

General experimental procedures:

Column chromatography was carried out using silica gel (70-230 mesh), and gel filtration column chromatography was carried out using sephadex LH-20. Fractions collected from column chromatography were monitored on TLC using alumina-backed sheets with visualization under UV (254nm), Ninhydrin reagent and 10% sulfuric acid in methanol.

UV spectra were recorded on Shimadzu, A10833981012SM spectrophotometer. IR spectra were recorded on Shimadzu, IR Affinity-1S, FTIR spectrophotometer. ¹H NMR, ¹³C NMR and Mass analysis were carried out by Sophisticated Analytical Instrumentation Facility (SAIF) at Punjab University.

Fractionation and isolation:

The TLC analysis of NIO-971 (A001) and NIO-971 (A002) showed a similar profile and hence both the extracts were combined. Approximately 29g of crude extract was partitioned using solvents of increasing polarity starting with petroleum ether (PE), chloroform (CHCl3), and n-butanol, to get PE (≈3.8g), CHCl3 (≈1.5g), n-BuOH (≈3g), and aqueous fraction (≈15g). The aqueous fraction was freeze-dried and later lyophilized until a dry powder was obtained. Methanol was then added and methanol soluble part was separated, combined, and concentrated on a rotary evaporator to obtain methanol soluble part (≈3.5g) and methanol insoluble part (≈6.6g).

Series of column chromatography of the crude extract on silica gel and sephadex LH-20 yielded 8 compounds. n-BuOH fraction was subjected to series of column chromatography and considering the TLC pattern, the most enriched fraction after every column was rechromatographed to get isolated compounds. In total five columns were run on n-BuOH fraction yielding compound AN-01 and compound AN-02. Similarly, chloroform fraction was subjected to column chromatography; in total 4 columns were run depending on the TLC profile, and compound AN-03 and compound AN-04 were obtained. Compound AN-05 and compound AN-06 were obtained from the petroleum ether fraction by performing a series of silica gel column chromatography. Compound AN-07 and compound AN-08 were obtained from the methanol soluble part of the aqueous fraction using gel filtration column chromatography. Since compounds AN-01 and AN-03 were obtained in minute quantities, adequate characterization could not be possible.

Antimicrobial Activity:

The antimicrobial activity was performed using the agar well diffusion method against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Wells were made using a sterilized cork borer at the center of each plate previously impregnated with 100µL bacterial suspension. 50µL of the Zoanthus fraction was added in the well using a micropipette. The plates were incubated at 37°C for 24hours. Streptomycin was maintained as a positive control. The experiment was performed in triplicates for each of the Zoanthus fraction used.

Total phenolic content:

The total phenolic content was determined using the Folin-Ciocalteu reagent method. 1ml of standard Gallic acid/ Zoanthus test solution was mixed with 2.5ml of 10% v/v Folin-Ciocalteu reagent and 2.5ml of 7.5% w/v sodium bicarbonate solution. The reaction mixture was then kept in dark at room temperature for 45mins. The absorbance was measured at 760nm using a UV-Visible spectrophotometer.

The standard calibration curve of Gallic acid was plotted with the concentration of Gallic acid on the X-axis and absorbance on the Y-axis. The result of total phenolic content was expressed in mg/g of Gallic acid equivalent (GAE).

Total flavonoid content:

The total flavonoid content was determined using the Aluminum chloride method. 2ml of standard Quercetin/ Zoanthus test solution was mixed with 2ml of 2% aluminum chloride. The reaction mixture was then kept in dark at room temperature for 1hour. The absorbance was measured at 415nm using a UV-visible spectrophotometer.

The standard calibration curve of Quercetin was plotted with the concentration of Quercetin on the X-axis and absorbance on the Y-axis. The result of total flavonoid content was expressed in mg/g of Quercetin equivalent.

RESULTS:

Compound AN-02:

It was obtained as light green solid (15mg), soluble in chloroform. UV (λmax): 241.5nm and 284nm; IR (cm-1): 3396.6, 2954.9, 2854.6, 1641.4, 1462.0, 1377.1; 1H NMR: 5.37, 3.56, 1.54, 1.09, 0.98, 0.96, 0.73, 0.73; 13C NMR: 71.9, 140.7, 121.8; Mass Spectrum: Molecular Formula: C₂₉H₅₀O, Molecular mass (m/z): 414.7, ESI-MS m/z: 437.4098 [M+Na]⁺, 395.4 [M-H20-H] ⁺.

Compound AN-04:

It was obtained as white solid (161.4mg), soluble in chloroform. UV (λmax): 404.5nm, 242.5nm and 274nm; IR (cm-1): 3423.6, 2933.7, 2848.8, 1463.9, 1045.4; ¹H NMR: 3.48, 5.34, 1.37, 0.76, 1.23, 0.84, 0.89, 0.98; ¹³C NMR: 71.8, 140.7, 121.7; Mass Spectrum: Molecular Formula: C₂₈H₄₈O, Molecular mass (m/z): 400.68, ESI-MS m/z: 401.62 [M+H]+, 423.60 [M+Na]+, 383.66 [M-H20+H]+, 385.64 [M-CH3]+, 367.65 [M-CH₃-H20]+, 255.60 [M-(side chain+H20)]+.

Compound AN-05:

It was obtained as white solid (mg), soluble in chloroform. UV (λmax): 243.5nm and 281.5nm; IR (cm-¹): 2916.3, 2848.8, 1462.0, 1732.0; ¹H NMR: 0.86, 1.28, 2.27, 5.3, 4.0; ¹³C NMR: 129.9, 129.7, 64.4, 174.0; Mass Spectrum: Molecular Formula: C₃₂H₆₀O₂, Molecular mass (m/z): 492.4, ESI-MS m/z: 493 [M+H] ⁺.

Compound AN-06:

It was obtained as green solid (12mg), soluble in chloroform. UV (λmax): 669nm, 612nm, 538.5nm, 508 nm, 475nm, 414nm, 274nm, 326nm and 243.5nm; IR (cm-1): 2918.3, 1460, 1737, 1217; Mass Spectrum: Molecular Formula: C₅₅H₇₄N₄O₅, Molecular mass (m/z): 871, ESI-MS m/z: 871.3[M+H] ⁺, 593.3[M-C₂₀H₃₈]⁺.

Compound AN-07:

It was obtained as white solid (787mg), soluble in water. UV (λmax): 282.5nm; IR (cm^-1): 2916, 1217.0; ¹H NMR: 3.66, 1.48, 1.27, 0.85; 13C NMR: 65.3, 31.1, 28.9, 13.8; Mass Spectrum: Molecular Formula: C₂₄H₅₀O, Molecular mass (m/z): 354.7, ESI-MS m/z: 355.4 [M+H]⁺.

Compound AN-08:

It was obtained as white solid (405.4mg), soluble in water. UV (λmax): 283.5nm; IR (cm-1): 3402.4, 1629.8, 1411.8; 1H NMR: 3.05, 3.37; 13C NMR: 35.9, 47.5; Mass Spectrum: Molecular Formula: C4H10Br2N2, Molecular mass (m/z): 244/246/248, ESI-MS m/z: 267/269/271[M+Na] ⁺; Fragment ions: m/z 79/81[Br].

DISCUSSION:

β-sitosterol (compound AN-02):

β-sitosterol (AN-02) isolated from the marine source is currently referred to as an “orphan phytosterol” due to its poorly explored cellular mechanism of action^24,25,26 but which is richly beneficial in therapeutics. The present work has allowed us to inspect the FTIR spectrum of this compound which shows a characteristic broad absorption band at 3396 cm^-1 indicating the presence of the hydroxyl group in the molecule. The bands at 2954-2854, 1641.42, and 1462-1377 cm^-1 correspond to the C-H stretching vibrations, double bond C=C and bending vibrations for isopropyl groups, respectively. In the ¹H NMR spectrum, a typical multiplet is observed at δH 3.56 (m) for H-3 attached to the hydroxyl group. This is also revealed from the peak observed at δC 71.9 in ¹³C NMR. Peaks at δH 5.37 (H-6) are attributed to the presence of a double bond in the molecule. The peaks in ¹³C NMR at δC 140.7 and 121.8 also confirm the double bond at position C5 and C6 respectively. Careful observation of the ¹H and ¹³C NMR spectra with several peaks observed in the shielded region shows that the molecule contains saturated CH, CH₂, and CH₃ groups in the molecule. Both ¹H and ¹³C NMR spectra obtained look characteristic of sterol type of compounds. Prominent peaks for the methyl groups are observed in the 1H NMR spectrum. Doublet at δH 0.73 showed the presence of two methyl groups at H-26, 27. The intense peaks at δH 1.54, 1.09, 0.98, and 0.96 are attributed to the methyl groups (H-19, H-18, H-21, and H-29).

On comparison of the spectra with the literature reports, the compound is confirmed as β-sitosterol. The ESI-MS spectrum showed peak at m/z 437.4 [M+Na]⁺ and m/z 395.4 [M-H20-H]⁺ for β-sitosterol (Fig. No.2) with molecular mass m/z 414 (C₂₉H₅₀O).

Fig. No.2 Structure of beta-sitosterol

24-methylcholesterol (compound AN-04):

From the IR spectrum of this steroid derivative²⁷,a band at 3423.6 cm^-1 was observed for the O-H bond vibration of the hydroxyl group. The out of plane C-H vibrations of the unsaturated part was observed at 800cm^-1. The band at 2933.7cm^-1 corresponded to the stretching vibration of the methyl (C-H) group. The bands of vibrations of methylenic groups were seen at 2848 cm^-1 (C-H) and 1463 cm^-1. The C-C vibration was shown as a weak band at 1045 cm^-1. At δH3.48 a multiplet is attributed to H-3 proton as seen in the ¹HNMR spectrum and its corresponding C-3 is seen at δC 71.82. The presence of a double bond in the molecule is confirmed due to the presence of a peak at δH 5.34 (H-6) and also due to the presence of peaks at δC 140.7 and 121.7 at position C5 and C6 respectively in the ¹³C NMR spectrum. The ¹H NMR spectrum showed the presence of six methyl signals, that appeared as two methyl singlets at δH 0.76 and δH 1.23, three methyl doublets at δH 0.84, 0.89 and 0.98, and a methyl triplet at δH 1.37. Apart from this, several peaks were observed for CH₃, CH₂ in the shielded region which are characteristic for sterol type of molecules. All the spectra along with the values reported on comparison with reported literature confirmed that the compound is 24-methylcholesterol also known as campesterol.

The ESI-MS spectrum showed peaks at m/z 401.62 [M+H] ⁺, and m/z 423.60 [M+Na] ⁺. Many fragments ion peaks such as [M-H20+H]⁺, [M-CH₃]⁺, [M-CH₃-H₂0]⁺, [M-(side chain+H₂0)]⁺ were evident at m/z values of 383.66, 385.64, 367.65, 255.60, respectively. The molecular mass of 24-methylcholesterol (Fig. No. 3) is m/z 400 for molecular formula C₂₈H₄₈O.

Fig. No.3 Structure of 24-methyl cholesterol

(17E,19E)-2-hydroxydotriaconta-17, 19-dienoic acid (compound AN-05):

The band at 2916.3 cm^-1, 2848.8 cm^-1, 1462.0 cm^-1 in the IR spectrum is for C-H stretching which indicated the presence of a long fatty chain. The band at 1732.0 cm^-1is due to the presence of C=O in the molecule. In the ¹H NMR spectrum peaks of the triplet at δH 0.86 for methyl (CH₃) group were observed. The peak at δH 1.28 is for the methylene chain (CH₂)_n of the molecule. The peak of the methylene group (-CH₂) present next to the double bond was seen at δH value of 2.27. The double bond CH gives multiplets at δH 5.3 and corresponding peaks at δC 129.9 and 129.7. The peak of carbonyl carbon at δC was observed at 174.0. The δH 4.0 (triplet) is observed for hydroxyl carbon (-CH-OH) and its δC 64.4 in ¹³C NMR but the position of a double bond could not be confirmed. The ESI-MS mass spectrum showed a peak at m/z value of 493 [M+H] ⁺ for the hydroxyl fatty acid [C32:2] having a molecular mass of 492.4 and molecular formula C₃₂H₆₀O₃. Thus, the compound is identified as (17E,19E)-2-hydroxydotriaconta-17,19-dienoic acid (Fig. No.4).

Fig. No.4 hydroxydotriaconta-17,19-dienoic acid

Pheophytin a (compound AN-06):

This potential anticancer compound pheophytin a²⁸, which is a chlorophyll-related molecule sans the central Mg+ ion²⁹, showed absorbance at wavelengths 669, 612, 538.5, 508, 475, 414, 274, 326, and 243.5nm. The IR spectrum showed bands at 2918.3 and 1460 cm^-1 indicative of methylenic (C-H) groups. The band at 1737 cm^-1 indicates that the compound contains a C=O bond. The band at 1217 cm^-1 is for the C-O stretching vibration. The ESI-MS spectrum of the compound gave the [M+H]⁺ ion at m/z 871. Due to the loss of the phytadiene (C₂₀H₃₈) group, a peak was also observed at m/z 593. Based on UV and mass spectra and on comparing the data available in the literature reports, the compound was identified as pheophytin a referred to as pigment of life³⁰ (Fig. No.5) having a molecular mass m/z 871.7 and molecular formula C₅₅H₇₄N₄O₅.

Fig. No.5 Structure of Pheophytin a

1-(dodecyloxy) dodecane (compound AN-07):

In the IR spectrum of this alkane hydrocarbon absorption bands at 2916 cm^-1and 2848 cm^-1 revealed characteristic of C-H stretching vibration and for saturated hydrocarbon chain. The absence of bands in the region 3320-3600 cm^-1 shows that there is an absence of OH/NH in the molecule. In the ¹H NMR spectrum triplet at δH 0.85 is for methyl group (-CH₂-CH₃). The intense peak at δH 1.27 is for long-chain methylene (-CH2-) n, δH 3.66 (m) is for -CH2O-. The corresponding ¹³C NMR values observed are δC 13.8 (CH₃), 28.9 (CH₂) _n, 65.3 (-CH2-O), 31.1(-O-CH2-CH2-). Based on the ¹H NMR and ¹³C NMR the compound looks to be dodecyl alcohol (carbon 12). But since the FTIR spectrum did not show OH stretching frequency, the molecule should be symmetrical with the ether group (C-O-C). In the IR spectrum, the band at 1217.08 cm^-1 indicated the presence of a C-O-C stretch. The molecular ion is observed at m/z 355.4 [M+H] + which confirmed the structure to be 1-(dodecyloxy) dodecane (Fig. No. 6).

Fig. No.6 Structure of 1-(dodecyloxy)dodecane

1, 2-bis (2-bromoethyl) hydrazine (compound AN-08):

In the ESI-MS spectrum cluster of isotopic molecular ions was observed at m/z 267/269/271 in the ratio 1:2:1. This indicated that there is the presence of two bromines in the molecule. The presence of bromine is also revealed from molecular ion peaks at m/z 79 and m/z 81 (Br⁷⁹Br⁸¹). Only two triplets were present in the ¹H NMR spectrum at δH 3.05 (-CH₂NH-) and δH 3.37 (-CH₂Br) indicative of ethyl group and their corresponding δC 35.9 (-CH₂Br) and 47.5 (-CH₂NH-) in the ¹³C NMR. In the IR spectrum, a band was seen at 3402.4 cm^-1 indicative of N-H stretch. A band at 1629.8cm^-1 was indicative of N-H bending vibration. A band at 1411.8 cm^-1was seen due to the presence of C-H bending vibrations. The molecular mass of the molecule is 244/246/248. Thus m/z 267/269/271 is for isolated isotopic molecular ion. From the above data, it is concluded that the structure is 1, 2-bis (2-bromoethyl) hydrazine (Fig. No.7).

Fig.No 7. 1,2 Bis(2-bromoethyl)hydrazine

Antimicrobial activity:

The test compounds under study were screened for antimicrobial activity against three clinical strains of bacteria; two gram-negative strains, i.e., E.coli and, P.aeruginosa as well as S.aureus which is a gram-positive bacterium. The test compounds did not show any significant activity. The negative control of organic solvents such as chloroform, petroleum ether, methanol, and n-butanol did not show any activity.

Estimation of total phenolic content and total flavonoid content:

Phenolic and flavonoid compounds are gaining the importance they deserve because they are responsible for scavenging free radicals, which have been implicated in a range of diseases like cancer, cardiovascular disorders and aging. Their antioxidant potential is attributed to their ability to donate hydrogen atoms to free radicals³¹. Total phenolic content was determined using the Folin-Ciocalteu method. The concentration of Gallic acid was estimated through the standard Gallic acid calibration curve and then Gallic acid equivalent present in various Z.sansibarius fractions was calculated. Total phenolic content was higher in the petroleum ether (PE) fraction (18.92mg/g GAE) than the methanol (MeOH) soluble part of the aqueous fraction (7.02mg/g GAE). The antioxidant potential of the organism directly corresponds to the total phenolic content. The number of flavonoid compounds present in the organism corresponds to the total flavonoid content. The concentration of quercetin was estimated through the standard quercetin calibration curve and then quercetin equivalent present in various Z.sansibarius fractions was calculated. Total flavonoid content was higher in the petroleum ether fraction (8.05mg/g QUE) than the methanol (MeOH) soluble part of the aqueous fraction (1.38mg/g QUE).

CONCLUSION:

Our study of zoanthids led us to characterize six out of eight isolated bioactive compounds- β-sitosterol (AN-02), campesterol (AN-04), (17E, 19E)-2-hydroxydotriaconta-17, 19-dienoic acid (AN-05) and pheophytin a (pigment) (AN-06), 1-(dodecyloxy) dodecane (AN-07) and 1,2Bis (2-bromoethyl) hydrazine (AN-08), each having its own beneficial property. Although Zoanthus sansibaricus extract did not exhibit any significant antimicrobial activity compared to the standard against three strains tested of bacteria; Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853). In addition, the reported total phenolic and total flavonoid content values have indicated that this marine organism can be explored for its antioxidant potential, thereby paving the way for the discovery for newer pharmaceutical leads. In the future, apart from antimicrobial activity, pure compounds can be screened for other pharmacological activities like antioxidants, cytotoxicity, anti-diabetic, etc. to assess their potency in treating various diseases. Our study placed Zoanthus sansibaricus under limelight in the hope that this generally overlooked organism may one day stand on the pedestal of lead compounds in drug discovery because every idea, every experiment and every study are stepping stones for the betterment of the pharmaceutical sector and therefore society and mankind.

ACKNOWLEDGEMENT:

A very special word of thanks to Dr. Sunil Kumar Singh, Director, CSIR-NIO, Goa, and Dr. Narsinh Thakur, Head of Chemical Oceanography Division, CSIR-NIO, Goa, for permitting us to undertake this research work at the CSIR-NIO institute. Our gratitude to Dr. Maria Pinto, Head of Microbiology Department, Goa Medical College and Dr. Yogita Sardessai, Head of Microbiology Department, Goa College of Pharmacy, for permitting us to carry out the antimicrobial activity. And in particular, we would like to acknowledge Sophisticated Analytical Instrumentation Facility (SAIF) at Punjab University for carrying out ¹H NMR, ¹³C NMR, and Mass analysis.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest that could potentially influence the reported work.

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Received on 25.11.2022 Modified on 18.07.2023

Research J. Pharm. and Tech 2024; 17(1):142-148.

DOI: 10.52711/0974-360X.2024.00023