Journal of ISSN: 2473-0831 JAPLR

Analytical & Pharmaceutical Research
Research Article
Volume 1 Issue 1 - 2015
Isolation, Characterization and Structural Elucidation of Bioactive Compound in Aurora Globostellata
Chairman K1, Ranjit Singh AJA2*, Murugesan AG3 and Vasanthi K4
1Department of Microbiology, Kamarajar Govt. Arts College MS University, India
2JP College of Arts and Science, India
3Department of Environmental Science, MS University, India
Received: April 9, 2015| Published: October 13, 2015
*Corresponding author: Ranjit Singh AJA, The Principal, JP College of Arts and Science, Ayikudi, Tenkasi, Tirunelvei, India, Email: ,
Citation: Chairman K, Ranjit SAJA, Murugesan AG, Vasanthi K (2015) Isolation, Characterization and Structural Elucidation of Bioactive Compound in Aurora Globostellata. J Anal Pharm Res 1(1): 00003.DOI: 10.15406/japlr.2015.01.00003


Marine environment is a repository of many valuable therapeutic substances. Of the different marine biota, the benthic community, Poriferans (Sponges) are goldmine for many pharmaceutical compounds. The sponges were identified using meristic and morphometric characteristics. The sponge species which have confusion in conventional taxonomical study were subjected to 18S rRNA genomic sequencing studies. The present study of sponges, the sponge Aurora globostellata was further selected for an in-depth study based on the bioactive potential and quantum of availability. The compound was crystallized and subjected to NMR (1H-NMR, 13C-NMR, DEPT-135, DOSY), HR-MS, IR, UV and fluorescent analysis. The structure of the compound in this fraction was characterized and it was identified as Azasterol glycoside.

Keywords: Aurora globostellata; 1H-NMR, 13C-NMR, DEPT-135, DOSY); HR-MS; IR; UV spectrometer


NMR: Nuclear Magnetic Resonance analysis; HR-MS: High Resolution Mass Spectrum; UV: Ultra Violet spectroscopy; IR: Infra Red spectroscopy; RP: Reversed Phase


The ocean covers about 70% of the earth surface providing a diverse living environment for invertebrates [1]. Therefore, marine natural products will play a major role in drug discovery in the future. The work on marine natural products started 59 years ago when Bergman discovered the novel bioactive Arabino-nucleoside from marine sponge Cryptotethya crypta [2]. This discovery encouraged natural products chemist to pay attention to marine natural products as important biomedical source. In order to survive in a highly competitive environment, marine invertebrates produce a tremendous diversity of extreme toxic compounds. This has stimulated research groups to screen marine samples in various cytotoxic assays.

Some of the compounds from marine invertebrates initially discovered were either too toxic or not effective in treating diseases for pharmaceutical purposes, but were found to be useful as biological tools or as cosmetic ingredients or as agrochemicals [3]. The Caribbean gorgonian, Pseudopterogorgia elisabethae, is an example of a source of marine natural product used in the cosmetic industry. The extract from this gorgonian shows anti-inflammatory activity, which nowadays is used as an ingredient in cosmetic skin care products [4]. Largest groups of marine invertebrates as a source of secondary metabolites are the sponges. The structurally diverse varieties of metabolites have high therapeutic potential to treat human diseases and have made them worthy of research for marine natural chemists [5]. Natural products isolated from the phylum Porifera account for 50 % of those reported from marine invertebrates. About 98 % of these metabolites are derived from amino acids, Acetogenin, and the Isoprenoid pathway [6].         

There are many classes of alkaloids which were isolated from marine sponges. However, one interesting group is the Bromopyrrole-imidazole alkaloids due to its biological activity and structural diversity. About 90 compounds of this class of alkaloids were characterized [7].

Marine organisms are rich sources of unconventional steroids. Numerous uncommon steroids have been discovered from a wide variety of marine sources, especially sponges, octocorallias, and echinoderms, since the early 1970s [8,9].

Ishibashi et al. [9] isolated 10 new 7-hydroxysterols hav­ing variations on the side chains from a sponge, Topsentia sp. collected in Okinawa. Kobayashi et al. [10] earlier de­scribed the discovery of three new C29 steroids, Xestokerols A-C, having a Cyclopropane ring on the side chain from an Okinawa sponge, Xestospongia sp.

Several reports describe isolations of new sulfated steroids with anti­microbial activity from several species of sponges. The compounds are Annasterol sulfate from the Pacific deep water sponge Poecillastra laminaris [11], Acanthosterol sulfates A-J fromAcanthodendrilla sp [12], Topsentiasterol sulfate A-E from Topsentia sp. [13], and echinoclasterol sulfate phenethyl ammonium salt from.

 A highly oxygenated 9, 11-secosterol, stellettasterol, has been reported as a constituent of the sponge Stelletta sp. [14]. Nitrogenous steroids or steroidal alkaloids are rather rare from marine sources [15]. Reported a steroid/amino acid conjugate, polymastiamide A, from the Norwegian sponge Polymastia boletiformis. The marine environment, which contains a vast array of organisms with unique biological properties, is one of the most underutilized biological resources. To date, algae and microalgae are referenced in the literature as sources of bioactive compounds for use as functional food ingredients [16,17]. In some of these matrices, interesting functional compounds were isolated previously [18-20].

Experimental Section

Collection of sponges, sample preparation and extraction

The sponges 22 species were collected from the low inter tidal pools at Bay of Bengal from Gulf of Manner Biosphere reserve of Tuticorin coast (8°47¢ N, 78° 8¢ E) and Rameswaram coast (9° 28¢ N, 79° 12¢ E) at 4-5 m depth from Tamil Nadu, India. The sponges were collected by an eco-friendly bulk collection by catch in the fishing nets. From the all twenty one sponges identified, tissues samples were incised out and (100g) were washed with sea water, air dried and chopped into small size before being ground into fine paste. Using the paste the Ethyl acetate (EtOAc) and Methanol (MeOH) were carried out. The extraction was carried out in triplicates for 48 h. the extract was stored in dark container and left in deep freezer at - 20° C. After 48 h the extract was filtered through Whatman filter paper (No: 2) and concentrated using vacuum rotary evaporator (Super fit, Bangalore).

Thin layer chromatography (TLC)

Each fractions of the column eluted sample was subjected to TLC to find out a single compound in the fraction. TCL was performed on a prepared plates with Si-gel F254 grade (Merck, Darmstadt, Germany) as stationary phase. Liquid mobile phases were either semi polar (CH2Cl2: MeOH; 9:1, v/v) or non polar (Hexane: EtOAc; 8: 2, v/v). Reversed phase (RP) was used for polar fractions. A one-dimensional ascending development technique was used to detect the constituents of an extract on TLC plate. Visual detection was done in daylight and under UV light at a wave length of 254 and 344 nm depending on the group of compounds investigated.

Column chromatography

So sponges are the rich source of novel bioactive compounds. Hence in the present study the column chromatographically eluted 16th fraction of ethyl acetate extracts of Aurora globostellatawas reported to exhibit a good antimicrobial and antiviral activity. Hence the eluted compound was crystallized and subjected to Nuclear Magnetic Resonance (NMR) analysis (1H-NMR, 13C-NMR, DEPT-135, DOSY-NMR), High Resolution Mass Spectrum (HR-MS) Ultra Violet spectroscopy (UV), Infra Red spectroscopy (IR) and fluorescence spectroscopy studies to elucidate the structure of the compound isolated from this fraction of column eluted sample.

Results and Discussion

Hexane: Ethyl acetate (8:2%) fraction yielded a homogenous compound. It is a greenish yellow crystal, melting points 274 to 276. It answered Liebermann and Burchard test and also Molisch test. It indicated that it may be steroidal glycosides. Elemental analysis indicated that Nitrogen (N) is present in the compound.

In TLC Hexane: Ethyl acetate (60:40), it gave a single pink colored spot. When methanolic sulfuric acid was sprayed and heated to 80°C in hot air oven for 5 minutes, the colour of the spot indicated the presence of Azasterol Glycoside.


Twenty five mg of compound was refluxed with 10 ml of 2 N HCl for 20 minutes. The condense were separated by separating funnel. 25 ml ethyl acetate was added shaken well. Aglycone part is in ethyl acetate fraction. The mass spectral data of Aglycone confirmed the presence of Azasitosterol (m/z 429) and b-Sitosterol (m/z 414).

The aqueous layer after concentration was subjected to paper chromatographic analysis in Whatman No: 1 filter paper with authentic sugars. The sugar moiety in the compound was identified as a disaccharide possibly b-D, Glucopyranosyl-glucopyranoside accounting the side chain mass spectrum of the compound (m/z 803). This further confirmed the presence of the Azasterol glycoside (Nitrogen substituted b- Sitosterol glycoside).The molecule formula of Aglycone part is C29 H51 NO. m/z peak 803 mass indicates the total part of compound structure. (Nuclear Magnetic Resonance (NMR) analysis (1H-NMR results Figure 1-5, 13C-NMR, Figure 6, DEPT-135, Figure 7 DOSY-NMR), High Resolution Mass Spectrum (HR-MS) Ultra Violet spectroscopy (UV), Infra Red spectroscopy (IR) and fluorescence spectroscopy)

Figure 1: 1H-NMR spectra of purified compound 1(Azasterol glycoside).
Figure 2: 1H-NMR spectra of purified compound 1(Azasterol glycoside).
Figure 3: 13C-NMR spectra of purified compound 1 (Azasterol glycoside).
Figure 4: DEPT-135 spectra of purified compound 1 (Azasterol glycoside).
Figure 5: HR-MS spectra of purified compound 1(Azasterol glycoside).
Figure 6: HR-MS spectra of purified compound 1(Azasterol glycoside).
Figure 7: DOSY spectra.

Aglycone part

The mass spectra also indicated that the compound has Azasterol glycoside. The m/z 414 indicated that the Aglycone part of the compound was b-Sitosterol. The molecular weight of glycone part was 374 leading to a disaccharide attachment possibly b-D Glucopyronosyl-glucopyranoside (Figure 8).

Figure 8: DOSY spectra.

High Resolution Mass Spectrum (HR-MS) (Figure 5)

M/Z peak at 803 (M+ peak) Molecular ion peaks.
M/Z peak at 804 (M+ 1 peak) Molecular ion peaks with one proton abstracted from solvent, and again one peak found at M/Z peak at 413 (Base peak – most stable peak).

IR Spectrum (Figure 6)

Peak at 2951 => aliphatic CH2 and CH3 groups
Peak at 1617 => C=C stretching and N-H bending can give rise to a band about 1600 cm-1
Peak at 1268 => C-C stretching and C-N- stretching can give rise to a band about 1260 cm-1
IR data suggest that the compound is accordance with a sterol derivative.

Calculation of Hydrodynamic Radii

Stoke-Einstein Equation rs
where rs is the hydrodynamic radii, k is the Boltzmann constant (1.3807 x 10-23 m2Kgs-2K-1), T is the temperature (298 K), η is the viscosity of the CHCl3 solvent (5.8 x 10-4 Kgm-1s-1) and D is the diffusion co-efficient (9.827 x 10-10 m2s-1).

The average diffusion co-efficient of the compound 1, Azasterol glycoside is obtained from the DOSY-NMR studies. It is clearly evident from the DOSY spectra (Figure 7) that the isolated compound is pure and a single compound is separated from the extract. The contours corresponding to the 1H-NMR chemical shifts are along a same horizontal line indicates the existence of single compound in the solution. The hydrodynamic radii calculated using Stoke-Einstein equation is 3.8 Ǻ. (Figure 7) DOSY-NMR spectra of compound 1 in CDCl3 at 298 K.

    •  UV-VIS and Fluorescence Studies

(Figure 9) depicts the uv-vis and fluorescence spectra of compound 1, Azasterol glycoside in Acetonitrile. The uv-vis spectra of compound 1 shows a maximum absorption at 275 nm which may be attributed to the n-π* or π-π* transition. A very dilute acetonitrile solution of compound 1 Azasterol glycoside upon excitation at 275 nm shows a strong fluorescence emission at 350 nm and excitation spectrum is the mirror image of the emission spectra. This study clearly reveals that the compound 1, Azasterol glycoside is highly fluorescent.

Figure 9: Depicts the uv-vis and fluorescence spectra of compound 1.

(Figure 10) UV-VIS spectra of compound 1, Azasterol glycoside in acetonitrile solution and b) emission and excitation spectra of compound 1 in acetonitrile solution. λexc =275 nm, scan rate = 300 nm/min, excitation slit width = 8.0 nm and emission slit width = 8.0 nm.

Figure 10: UV-VIS spectra of compound 1.

The biological evaluation of marine derived extracts and pure compounds for pharmaceutical development have been based on assays development from the libraries of the already developed synthetic compounds. Marine microbes as model systems offer the potential to understand and develop treatments of disease based on the normal physiological role of their secondary metabolites. For example, the mechanisms of toxin action are well-known and are currently being applied to the development of new drugs.


From the current study it could be noted down that a vast stretch of bioactive compounds present in the marine environment is still unexplored. The bioactive compound Azasterol glycoside from Aurora globostellata which were found to be bioactive compound. Also these active factors could be used to develop antimicrobial wound care finishes which could replace the commercial wound care agents, being cheaper in cost and effective in action.


Authors are thankful to Principal, Kamarajar Govt. Arts and Science College, Surandai, M.S.University, Tirunelveli– 627 859, Tamilnadu, India, lab facilities and encouragement. We thankful for supporting agency of University Grant Commission, Govt. of India, New Delhi.


  1. Lalli CM, Parson TR (1993) Biological Oceanography. New York, USA, pp. 1-10.
  2. Bergmann W, Freeney RJ (1951) Contribution to the study of marine product of marine products. The nucleosides of sponges. I J org chem 16(16): 981-987.
  3. Fenical W (1997) New pharmaceuticals from marine organism. Trends biotechnol 15(9): 339-341.  
  4. Proksch P, R Edrada RA, Ebel R (2002) Drugs from the seas-current status and microbiological implications Appl. Appl Microbial Biotechnol 59(2-3): 125-134.
  5. Ireland CM, Copp BR, Forster MP, McDonald LA, Radisky DC (1993) Biomedical potential of marine natural product. In: Attaway D & Zaborsky OR (Eds.), Pharmaceutical and bioactive natural products. Marine biotechnology. Plenum publishing Corp, New York, USA, pp. 1-43.
  6. Hooper JNA, Van Soest RWM (2002) The systema porifera-A Guide to the classification of sponges. Kluwer Academic /Plenum publishers, New York, pp. 1-1756.
  7. Hoffmann H, Lindel T (2003) Synthesis of the pyrrole-imidazole alkaloids. Synthesis 12: 1753-1783.
  8. Schmitz FJ (1978) Uncommon marine steroids. Marine Natural Products, Chemical and Biological Perspectives. Academic Press 1: 241-297.
  9. Ishibashi M, Yamagishi E, Kobayashi J (1997) Topentinols A-J New sterols with highly branched side chains from marine sponge Topsentia sp. Chem Pharm Bull 45: 1435-1438.
  10. Kobayashi J, Cheng JF, Ishibashi M, Walchli MR, Yamamura S, et al. (1991) Two novel azetidine alkaloids with potent actomyosin ATPase activating activity from the Okinawan marine sponge Penares sp. J Chem Soc Perkin Trans 1: 1135-1138.
  11. Makarieva TN, Stonik VA, D’yachuk, Dmitrenok S (1995) Annasterol sulfate, a novel marine sulfated steroid, inhibitor of glucanase activity from the deep water sponge Poecillastra laminaris. Tetrahedron Lett 36(1): 129-132.
  12. Tuskamoto S, Kawabata T, Kato H, Ohita T, et al. (2007) Naamidines 4 and 1 cytotoxic Imidazole Alkaloids from the Indonesia marine sponge Leucetta chagosensus. J Nat pro52: 4181-4186.
  13. Fuestani N, Sugawara T, Matsunago S (1992) Bioactive marine metabolites 41 Theopederins A-E, potent antitumor metabolites from a marine sponge Theonella sp. J Org Chem 57(14): 3828-3832.
  14. Li H, Matshunaga S, Fusetani N (1994) A new 9, 11 - secosterol, stellettasterol from a marine sponge Stellatta sp. Experientia 50(8): 771-773.
  15. Kong F, Andersen RJ (1993) Polymastimide A, a novel steroid/amino acid conjugate isolated from the Northwegian marine sponge Polymastia boletiformis (Lamarck 1815). J Org Chem 58(24): 6924-6927.
  16. Plaza M, Santoyo S, Jaime L, Garcia-Blairsy G, Herrero M, et al. (2010a) Screening for bioactive compounds from algae. J Pharm Biomed Anal 51(2): 450-455.
  17. Plaza M, Amigo-Benavent M, Castillo MD, Ibanez E, Herrero M (2010b) Facts about the formation of new antioxidants in natural samples after subcritical water extraction. Food Research International 43(10): 2341-2348.
  18. Kadam SU, Prabhasankar P (2010) Marine foods as functional ingredients in bakery and pasta products. Food Research International 43(8): 1975-1980.
  19. Kim SK, Wijesekara I (2010) Development and biological activities of marine-derived bioactive peptides: a review. Journal of Functional Foods 2(1): 1-9.
  20. Parekh J, Chanda SV (2007) In vitro antimicrobial activity and Phytochemical analysis of some Indian medicinal plants. Turk J Biol 31: 53-58.
© 2014-2016 MedCrave Group, All rights reserved. No part of this content may be reproduced or transmitted in any form or by any means as per the standard guidelines of fair use.
Creative Commons License Open Access by MedCrave Group is licensed under a Creative Commons Attribution 4.0 International License.
Based on a work at
Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version | Opera |Privacy Policy