Analysis of Bioactive Compounds from Methanol Extract of
Diadema setosum Sea Urchin Gonads using Gas Chromatography –

Mass Spectrometry

 

Silvester Maximus Tulandi*, Lisawati Tanzil, Dian Maria Ulfa

Department of Pharmaceutical and Food Analysis, Health Polytechnic Jakarta II,

Ministry of Health, Jakarta, Indonesia.

 

ABSTRACT:

This study aimed to determine the bioactive component of gonads of the sea urchins Diadema setosum from Indonesian waters using Gas Chromatography Mass Spectrometry. Phytochemical test results qualitatively showed that the methanol extract of D. setosum gonads contained steroids-triterpenoids, saponins and alkaloids while the results of analysis using GC-MS illustrated that the largest contents were Hexadecanoic acid, methyl ester (29.53%) and Tetradecanoic acid, methyl ester (14.86%), followed by other components in small quantity including Methyl 13-methyltetradecanoate (0.55%), Pentadecanoic acid, methyl ester ss Methyl n-pentadecanoate (2.08%), Hexadecadienoic acid, methyl ester (0.48%), 9-Hexadecenoic acid, methyl ester (4.68%), Methyl 10-methyl-hexadecanoate (0.56%), Hexadecanoic acid (4.05%), Heptadecanoic acid, Methyl ester (4.17%), 6,9,12-Octadecatrienoic acid, Methyl ester (1.33%), 9-Octadecenoic acid (Z)-, Methyl ester (5.42%), Octadecanoic acid, Methyl ester (1.91%), 9,12-Octadecadienoic acid (Z, Z) -, Lineloic acid (8.58%) , 5,8,11,14-Eicosatetraenoic acid, Methyl ester (3.21%), 9,12-Octadecadienoic acid, Methyl ester (4.71%), Cis-11-Eicosenoic acid, Methyl ester (1.86%), (1S, 15S) -Byciclo [13.10] hexadecan-2-one (4.82%), Cholesta -3.5-diene (0.67%), Cholest-5-en-3-ol (3β) - (4.20%). Components of the compound were reported to possess pharmacological function as antimicrobial, antifungal, antioxidant, anticancer, hipercholesterolemic agent, lubricant, antiinflammatory, nematicide, hepatoprotective, antihistaminic, pesticide, larvacidal activities, antiacne, anemiagenic, antiandrogenic, 5-alpha reduce inhibitor, antiarthritic and anticoronary properties.

 

 

 

INTRODUCTION:

Utilization of natural ingredients as alternative medicine in the world of health has recently increased rapidly. Increased exploitation of biological activities from various medicinal ingredients from natural ingredients has been carried out by many researchers because they are considered to be cost effective and having low side effects compared to treatment using synthetic chemicals^1,2. World Health Organization (WHO) estimates that around 85-90% of the world's population consume medicines that come from nature³.

 

Drugs originating from nature are safer than synthetic drugs because they are formed through natural biochemical pathways in organisms producing the components in the form of metabolism. Natural medicine has been used all over the world for the treatment and prevention of various diseases, especially in developing countries such as Indonesia where infectious diseases are endemic and the modern health facility is inadequate modern health. This has encouraged the researchers to correlate phytochemical elements from plants, animals, microorganisms on land and sea to study their pharmacological activities^4,5,6.

 

The Ocean is considered to be a source of potential drugs and some of these bioactive compounds or secondary metabolities have biomedical potential⁷. Sea urchin is a marine invertebrate. The experts classify sea urchin to be included in Echinoidea Class, Echinoderms Phylum. This organism is very abundant, known about 950 species in the world, while there are about 84 types of sea urchins in Indoensia⁸.

 

Echinoderms, have poisonous thorns on the surface of their skin. Sea urchin is noctural animal, which is active at night to find food, and hidden in crevices of coral during the day. Brown algae, green algae and seagrasse are common foods for sea urchin⁹.

 

Bioactivity studies of Diadema setosum sea urchin gonads indicate antioxidant activity¹⁰. Besides the sea urchin gonads from the Anthocidaris crassipina species contains astaxanthin compounds, which have antioxidant property¹¹. Salmacis virgilata sea urchin from Mudasalodai waters, India, can be used as antimicrobial and antioxidant. Antioxidant activity obtained from this type of sea urchin is better than ascorbic acid¹². Psammechinus milliaris sea urchins gonad from Loch Creran waters, Scotland (UK) contains many unsaturated fatty acids, omega-3, omega-6, EPA and DHA¹³. These studies confirm that sea urchin has the potential in disease prevention and treatment. However, research in Indonesia about the potential originating from the type of Diadema setosum sea urchin is still less done. Utilization of sea urchin in Indonesia is currently limited only as additional livestock feed and as a side dish for a small portion of coastal communities that are categorized as poor. Sea urchin is often regarded as a nuisance for beach tourism due to their poisonous spines and eating seaweed that is cultivated by fishermen⁹. This type of sea urchin is found in the waters of Kepulauan Seribu. The abundant population of Diadema setosum sea urchin in the waters of Indonesia also has the potential in the prevention and treatment of diseases underlying this research. The information of the research about the bioactive components of extracts derived from Diadema setosum sea urchin gonads hopefully can be a reference in subsequent studies.

 

The purpose of this study was to analyze the bioactive components of Diadema setosum sea urchin gonads crude extract in the waters of Kepulauan Seribu, Jakarta, Indonesia.

 

MATERIAL AND METHODS:

Sample Collection:

The samples used in this study were collected from the waters of Kelapa Island of Kepulauan Seribu (5^o39’11.1”S 106^o34’03.9”E) Jakarta - Indonesia. These samples were then identified macroscopically by looking at the shape, color and specific characteristics found in the sea urchin. Determination was carried out at the Research Center for Oceanographic, Indonesian Institute of Sciences.

 

 

Preparation of Extract:

Fresh Diadema setosum gonads was weighed as many as 50grams, cut into small pieces, then grinded. Maceration used methanol as a solvent in the ratio of 1:3 w/v to the mass of the sample, in a tightly closed glass bottle. They were shaked firmly and leaved for a day and night. The methanol layer was separated, and remassed until the methanol layer was colorless. Collection of methanol layer was filtered. A collection of methanol filtrate was concentrated with rotary vaccum at 40°C until a half-dry mass was obtained, then it was flown nitrogen until a dry extract was obtained. The extract was stored at 4°C before analysis.

 

Preliminary phytochemical screening:^14,15

Phytochemical analysis was conducted to determine the type of secondary metabolite compounds in the sea urchin gonads qualitatively. Compound tested included falvonoids, alkaloids, phenol hydroquinines, and tannins.

 

Steroid Test:

A total of 1mg of extract from each fraction was dissolved in 2mL of DMSO and put in a test tube. 10 drops of anhydrous acetic acid and 3 drops of sulfuric acid P were added to the mixture. The test results were positive if the solution formed red and turned blue and green.

 

Flavonoid Test:

One mg of extract from each fraction was added 0.1mg of magnesium powder, 0.4mL of amyl alcohol and 4mL of alcohol, then shaken. The test results were positive if they were red, yellow or orange color on the amyl alcohol layer.

 

Alkaloid Test:

A total of 1mg extract of each fraction was dissolved in a few drops of 2 N sulfuric acid, after which it would be tested with several alkaloid reagents including Dragendorff, Meyer, and Wagner. The test results were positive if brown deposits were formed for Wagner reagents, yellowish-white deposits for Meyer reagents, and orange-red deposits for Dragendorff solvent.

 

Saponin Test:

A total of 1mg sample was dissolved in hot water and then shaken until it would produce foam. Positive results obtained if the sample produced foam that was stable for 30 minutes and did not disappear if added 1 drop of HCl 2 N.

 

Phenol Hydroquinone Test:

A total of 1mg sample from each extract fraction was added with 20mL DMSO. The amount of 1mL of the fraction solution was taken then added 2 drops of 5% FeCl₃ solution. Positive results obtained if the extract fraction solution was formed in green or blue green.

Tannin Test:

A total of 1g sample was added with 3% FeCl₃ reagent. The formation of dark blue showed positive results.

 

Component Analysis using Gas Chromatography-Mass Spectrometry:

The component analysis of the compounds in the extract was carried out using GC-MS. The number of compounds contained in the extract was indicated by the number of peaks on the chromatogram, while the names/types of compounds interpreted were based on spectral data from each of these peaks using the library approach method in the GC-MS database and mass spectra data.

 

About 2μL methanol extract was injected into the GC-MS Instrument. GC-MS used in this analysis was GC-MS Agilent Technologie, with HP-5MS column type, 60 m column length, 0.25mmID column diameter, with temperature limit from 80°C – 300°C with temperature increase of 10°C/minute, flow rate of 1 mL/minute, oven temperature of 70°C – 290°C, interface temperature of 280°C, Split Ratio of 50.0, ion source of 200°C and Helium as carrier gas. The resulting data was matched with the compounds contained in the GC-MS database.

 

RESULTS AND DISCUSSION: 

Identification of Diadema setosum Sea Urchin:

The sea urchin used is black with black spines extending upwards, while at the bottom it is rather short. Has 5 white dots at the top and is located between segments of every 1 white dot, there is an orange anal sac (indicated by arrows) as a marker of the Diadema setosum species as shown in (Fig. 1).

 

Fig 1. A. Diadema setosum Sea Urchin; B. Anal sac is orange, the marker of Diadema setosum

 

Preparation of extract:

The gonads extract of Diadema setosum sea urchin was carried out by remaseration. Remaseration was done by immersing 50grams of wet gonad that have been grinded first using a methanol solution as many as 3 times the weight of the material, the gonad soaked was shaken for 30 minutes then allowed to stand for 24 hours, then the methanol layer was separated. Remaseration was carried out until the methanol layer was colorless, then filtered. The filtrate was evaporated in a rotary evaporator at     40 ⁰C until the extract mass was semi-solid. The extract obtained was then frozen with nitrogen gas. The gonads extract of Diadema setosum sea urchin obtained as much as 19.53gr. The yield of methanol extract was 39.06%.

 

The extraction process in this study used methanol as a solvent because methanol is a solvent from the good alcohol group used for preliminary extraction due to its ability to extract the active components. Methanol solvents are able to extract components derived from alkaloids, phenolic, carotenoids, tannins, sugars, amino acids and glycosides. In addition, methanol solvent also has less polar property compared to water, thus methanol solvent is able to destroy cell walls and causes the components in the cell to disintegrate and dissolve in the methanol solvent¹⁶.

 

Preliminary phytochemical screening:

Phytochemical screening of methanol extract from D. setosum sea urchin gonads was carried out qualitatively by the color reaction and deposition test method. The test was carried out on alkaloids, flavonoids, phenol-hydroquinone, steroids/triterpenoids, tannins and saponins. The test results showed that the methanol extract of Diadema setosum sea urchin gonads contained alkaloids, steroids/triterpenoids, and saponins. The results are reported in the (Table 1).

 

Table 1. Chemical compound analysis results of Diadema setosum sea urchin gonads

Explanation: (-) = not detected; (+) = detected

 

GC-MS Analysis:

Gas chromatography-mass spectrometry (GC-MS) is a useful tool for analysis of a wide range of relatively volatile compounds, and the technique has been widely applied in medical, biological, and food research¹⁷. GC-MS analysis was performed on crude methanol extract from D. setosum sea urchin gonads, an advanced step to identify bioactive compounds contained in the extract. Interpretation of the GC-MS mass spectrum was carried out using the National Institute of Standards and Technology (NIST) database and the Wiley library. The spectrum of unknown components was compared with the spectrum of components stored in the NIST database and the Wiley library to ascertain the molecular name, molecular weight and structure of the identified components.

 

Fig 2. The gas-chromatogram of the methanol extract of Diadema setosum gonads. 

 

The results of identification with GC-MS on methanol extract of Diadema setosum sea urchin gonads with a range of 92-99% similarity. The results are reported in the (Table 2). Literature study results on the pharmacological activity of each components as shown in (Tabel 3). From the results of the GC-MS analysis, it was found that the largest contents in Diadema setosum gonads crude extract were Hexadecanoic acid, methyl ester (29.53%), and Tetradecanoic acid, methyl ester (14.86%) which were seen in each peak area. Other components identified in small amounts were as follows: Methyl 13-methyltetradecanoate (0.55%), Pentadecanoic acid, methyl ester ss Methyl n-pentadecanoate (2.08%), Hexadecadienoic acid, methyl ester (0.48%), 9-Hexadecenoic acid, Methyl ester ss methyl ester (4.68%), Methyl 10-methyl-hexadecanoate (0.56%), Hexadecanoic acid (4.05%), Heptadecanoic acid, Methyl ester (4.17%), 6,9,12-Octadecatrienoic acid, Methyl ester (1.33%), 9-Octadecenoic acid (Z) -, Methyl ester (5.42%), Octadecanoic acid, Methyl ester (1.91%), 9,12-Octadecadienoic acid (Z, Z) -, Lineloic acid (8.58%), 5,8,11,14-Eicosatetraenoic acid, Methyl ester (3.21%), 9,12-Octadecadienoic acid, Methyl ester (4.71%), Cis-11-Eicosenoic acid, Methyl ester (1.86%), (1S, 15S) -Byciclo [13.10] Hexadecan-2-one (4.82%), Cholesta -3.5-diene (0.67%), Cholest-5-en-3-ol (3β) - (4.20%).

 

 

Table 2 GC-MS analysis result of methanol extract from Diadema setosum sea urchin gonads

 

Table 3. Pharmacological activity of components in the Diadema setosum gonads extract

 

Components of the compound were reported to possess pharmacological function as antimicrobial, antifungal, antioxidant, anticancer, hipercholesterolemic agent, lubricant, antiinflammatory, nematicide, hepatoprotective, antihistaminic, pesticide, larvacidal activities, antiacne, anemiagenic, antiandrogenic, 5-alpha reduce inhibitor, antiarthritic and anticoronary properties.

 

CONCLUSION:

This research helped to predict the content and structure of biomolecular compounds found in crude extracts of methanol from Diadema setosum sea urchin gonads, which can be used as medicine. Information about the bioactive compounds contained in the Diadema setosum gonads is expected to be beneficial for the development of natural medicines derived from the sea, and hopefully it can increase the economic value of this type of sea urchin. Isolation of bioactive components having pharmacological activities from these marine invertebrates is needed to provide benefits to the world of health.

 

ACKNOWLEDGEMENT:  

Financial support from the Agency for Development and Empowerment of Health Professionals, Republic of Indonesia Ministry of Health. The authors would wish to acknowledge the Director and Head of Research Management of Health Polytechnic Jakarta II, Ministry of Health for providing research facilities and encouragement.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 24.04.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1629-1634.