Rapid discrimination of china sponges by Tri-step infrared spectroscopy: A preliminary study

Journal of Molecular Structure xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc

Rapid discrimination of china sponges by Tri-step infrared spectroscopy: A preliminary study Jian-Hong Gan a,b,c, Chang-Hua Xu b,c, Hao-Bing Yu a, Hou-Wen Lin a,d,⇑, Qun Zhou c, Su-Qin Sun c,⇑ a

Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, PR China College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, PR China c Department of Chemistry, Tsinghua University, Beijing 100084, PR China d Research Center for Marine Drugs, Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, PR China b

h i g h l i g h t s  Nine sponges from two classes and six orders were discriminated by Tri-step IR.  Sponges of the same genus were definitely discriminated.  2DCOS-IR was applied to identify sponges with similar chemical profile distinctly.

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Sponge Discrimination Identification Infrared spectroscopy Two-dimensional correlation infrared spectroscopy

a b s t r a c t Trip-step infrared spectroscopy, Fourier transform infrared spectroscopy combined with second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR), was employed to characterize and discriminate nine China sponges. Sponges from different classes and different orders had respective unique IR macro-fingerprints. Their IR spectra suggested that the prime ingredient of calcareous sponges was calcium carbonate in calcite and/or aragonite forms, but that of demosponges was protein. Particularly, the sponges from the same genus which could not be identified by traditional spicule identification have been definitely discriminated. For sponges having highly similar chemical profile (IR spectral profile), SD-IR and 2DCOS-IR have been applied to enhance the spectral resolution to distinguish the sponges convincingly. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction There are 3 classes (Calcarea, Hexactinellida and Demospongiae), 25 orders, 127 families and 700 genera of marine sponges currently accepted in the living fauna with around 8500 named species reported worldwide [1–3]. Among them, more than 100 species of sponges in China are currently recorded. For identifying sponges, the prevalent traditional methods based upon skeletal structure (e.g. spicule methods) generally need a complete piece of sponge and heavily rely on experts mastering English, French and German and ages of experience. Today the number of experts in the sponge identification has been badly reduced. Though nonskeletal characters such as mode of reproduction, cellular characters, larval morphology have been used to identify sponges, the current classification is as complex as the diversity of sponges ⇑ Corresponding authors. Address: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, PR China. Tel./fax: +86 21 65585154 (H.-W. Lin). Tel.: +86 10 62781689; fax: +86 10 62782485 (S.-Q. Sun). E-mail addresses: [email protected] (H.-W. Lin), [email protected] (S.-Q. Sun).

themselves [4]. Therefore, it is essential to develop a new promising discrimination method which is more objective, rapid and simple. The modern infrared spectroscopy (FT-IR) is a rapid and direct method with high signal-to-noise ratio and good repeatability to analyze the complicated mixture systems such as food, Chinese herbal medicine [5]. Second derivative infrared spectroscopy (SDIR) can be used to handle severely overlapped spectra and enhance the apparent resolution [6]. If the differences in FT-IR and SD-IR spectra are too small to tell, two-dimensional correlation infrared spectroscopy (2DCOS-IR) can be employed to unfold FT-IR spectra in a second dimension to identify the differences more remarkably and convincingly [5]. With the holistic analytical method, ‘‘Tri-step infrared spectroscopy’’ (FT-IR combined with SD-IR and 2DCOS-IR), the extensive and exact analysis and identification of complicated mixture systems can be achieved [7,8]. In this article, nine different sponges of different classes, different orders and different species collected from Xisha islands of South China Sea have been investigated by using the Tri-step infrared spectroscopy to preliminarily establish a new method to identify sponges in a more objective, simpler and time-saving manner.

0022-2860/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molstruc.2013.12.088

Please cite this article in press as: J.-H. Gan et al., J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2013.12.088

2

J.-H. Gan et al. / Journal of Molecular Structure xxx (2014) xxx–xxx

2. Experimental

3. Results and discussion

2.1. Apparatus

3.1. Discrimination of sponges from different classes and orders

Spectrum GX FT-IR spectrometer (PerkinElmer, UK), equipped with a deuterated triglycine sulfate (DTGS) detector, in the range of 4000–400 cm 1 with a resolution of 4 cm 1. Spectra were recorded from an accumulation of 32 scans, and 0.2 cm/s 1 of OPD speed. The interferences of H2O and CO2 were subtracted when scanning. A CKW-II programmable temperature controller (Beijing Chaoyang Automatic Instrument Co., China) was arranged to perform the thermal perturbation in the range of 50–120 °C. Spectra were collected at each interval of 10 °C. The second derivative IR spectra were obtained after Savitzky–Golay polynomial fitting (13-point smoothing). Twodimensional IR correlation spectra were gained by using 2DCOS-IR correlation analysis software (designed by IR Lab, Tsinghua University) to analyze the series of thermoperturbation dynamic spectra.

Sponges are natural complicated systems. Their IR spectra represent a total accumulative absorption of all compositions and thus can provide the original whole chemical image (termed IR macro-fingerprint [9]) of sponges. As spectral peaks for the same particular function group in different molecules locate at the same spectral region with a small variation in wavenumber, the common information for a class of chemical compounds with similar molecular structures can be acquired [5]. When contents of chemical components vary, corresponding changes would occur to the peak profile of IR spectra. Fig. 2 shows the IR spectra of six species of sponges belonged to two classes. Different from the other five

1656 2961 2929

Aplysinopsis sp.

3417

1233 1320

1536 1454

Acanthella sp. 1 3329

Absorbance

1237

855 712 699 875 1082

1114

856 877 801713

2852

788

1648

2929

Aaptos aaptos

875

1639

3291

Plakortis simplex

875

1418 1647

874

Leucandra sp. 1803

1083 715

2523

2.3. Procedure 4000.0

Sponge samples were dried in vacuo and then were pulverized before IR measurement. Each sample (about 1–2 mg) was blended with KBr (100 mg), grounded into powder (200 mesh), and then pressed into a tablet.

3000

2000

1500

Aplysinopsis

Halichondrida

Axinellidae

Acanthella

Aplysinopsis sp. (AP)

Acanthella sp.1

Demospongiae

(AC1)

Acanthella sp.2 (AC2)

Suberitidae

Aaptos

Aaptos aaptos (AA)

China Sponges

Agelas clathrodes (AG1)

Agelasidae

Agelas

Agelas sp.2 (AG2)

Agelas sp.3 (AG3)

Hexactinellida Hamosclerophorida

Calcarea

Leucosolenida

400.0

Fig. 2. IR spectra of six sponges from different classes and orders: Aplysinopsis sp. (AP), Acanthella sp. 1 (AC1), Agelas clathrodes (AG1), Aaptos aaptos (AA), Plakortis simplex (PL) and Leucandra sp. (LE).

Spongiidae

Agelasida

1000

Wavenumber/cm-1

Dictyoceratida

Hadromerida

855

1534 14511404 1236 1081 1156

29612926

2930

475

1547 1409 1237 10801044 1450 1323

2962

3395

471

1045

1411 1443

Agelas clathrodes 3289

1655 1540

2924

1082

1649

2925 2854

2.2. Samples and reagents Sponges (Fig. 1) were collected from Xisha islands of South China Sea in April, 2007 and 2009 (Stored in Department of Pharmacy, Changzheng Hospital, Second Military Medical University) and was authenticated by Prof. Jinhe Li, Qingdao Institute of Oceanology, Chinese academy of sciences. KBr was purchased from Sigma (St. Louis, MO, USA).

1528 14501422

3300

Plakinidae

Grantiidae

Plakortis

Plakortis simplex

Leucandra

Leucandra sp.

(PL)

(LE)

Fig. 1. China sponges collected from Xisha Islands of South China Sea.

Please cite this article in press as: J.-H. Gan et al., J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2013.12.088

3

J.-H. Gan et al. / Journal of Molecular Structure xxx (2014) xxx–xxx Table 1 Preliminary assignment of main peaks in FT-IR spectra of six sponges.

Proteins

Calcite

Aragonite

Compounds with OH or/and NH groups Compounds with CH2 or/and CH3 groups

Band (cm 1) and main attribution

Sponge

1600–1690 (Amide I) 1480–1575 (Amide II) [10] 2515 1800 1420 875 713 2522 1788 1473 1080 855 713 700 3500–3200 (m(OH, NH))

1656 1528

Aplysinopsis sp. (AP)

Acanthella sp. 1 (AC1) 1649 1536

1422 875 712

877 713

1082 855 712 699 3300

1082 856 713 699 3417

2961 (m (CH3))

2961

2929, 2854 (m (CH2)) 1440-1555 (d(CH2, CH3))

2929 1450

2925, 2854 1454

demosponges (Class demospongiae), Leucandra sp. is a calcareous sponge and is collected for the first time in China. Preliminarily assignment of main peaks in the spectra is summarized in Table 1. The macro-fingerprint of the calcareous sponge is generally distinct from the other five demosponges. The demosponges have proteins as the common main compositions with two strong characteristic absorption bands in their IR spectra: amide I (1650 cm 1) and amide II (1535 cm 1), whereas the calcareous sponge contains a dominant composition of CaCO3 with less amount of proteins. Compared with the reference standards of two polymorphs of CaCO3 (Fig. 3) with Group-peak matching technique [9], as the calcareous sponge has five peaks at 2523, 1803, 1418, 874, 715 cm 1, one can know that the sponge mainly contain calcite (CaCO3). Based on group-peak matching technique, among the IR spectra of the five demosponges from different orders, AP has peaks at 1422, 875, 712 cm 1 and 1082, 855, 712, 699 cm 1 suggesting that AP contains calcium carbonate of both calcite and aragonite forms. Likewise, AC1 also contains the two forms of calcium carbonate but with lower contents. Only 3 (1080, 875 and 855 cm 1), 2 (1081 and 875 cm 1) and 0 peaks attributed to CaCO3 can be observed in AA, PL and AG1 spectra, respectively. In order to identify differences among the sponges more convincingly, SD-IR spectra were applied to enhance the spectral resolution. As shown in Fig. 4, overlapped peaks in the FT-IR spectra (Fig. 2) have been split into two or more peaks in SD-IR spectra

1424

Absorbance

Calcite

876 2513

1798

712

1473

Aragonite 855 1080 1788

712

Agelas clathrodes (AG2)

Aaptos aaptos (AA)

Plakortis simplex (PL)

Leucandra sp. (LE)

1655 1540

1648 1547

1639 1534

1647

3329

2924, 2852 1443

2523 1803 1418 874 715

875

875

1080 855

1081

1083

3289

3291

3395

2962

2961

2929 1450

2926 1451

2930

Aplysinopsis sp. 1499 1513 1450

1500 1515

Absorbance

Composition

1511

1468

1468

1411

1500 1453 1514 1468

1499 1403 1453 1512

1236

1500

1400

1106 1060 1081 1025

1323

Plakortis simplex

855

712 699

712

698

875

1075 1043

Aaptos aaptos

855

875

Agelas clathrodes

1081

874 855

855

1043

715 698673 654

715

598

698

875

1081 1043

875 855

715 698

Leucandra sp. 846

1418

1600.0

1082

Acanthella sp. 1

1081

1300

1200

1100

715

875

1000

900

800

700

550.0

Wavenumber/cm-1 Fig. 4. Second derivative IR spectra of six sponges from different classes and orders: Aplysinopsis sp. (AP), Acanthella sp. 1 (AC1), Agelas clathrodes (AG1), Aaptos aaptos (AA), Plakortis simplex (PL) and Leucandra sp. (LE).

disclosing more evident differences among the sponges. It can also be noted that more corresponding peaks (1418, 1081, 875, 855, 712, and 699 cm 1) to calcium carbonate can be observed for all sponges, verifying that all sponges contain calcium carbonate. Specifically, 5 (1081, 875, 855, 715 and 698 cm 1), 5 (1081, 875, 855, 715 and 698 cm 1) and 4 (874, 855, 715 and 698 cm 1) peaks attributed to CaCO3 can be observed in AA, PL and AG1 spectra, respectively. From above results, the sequence of relative content of CaCO3 can thus be deduced: AP > AC1 > AA  PL > AG1. Therefore, based upon the IR macro-fingerprints, the five demosponges from different five orders can be identified and distinguished straightforward.

700

2520

4000

3000

2000

1000

3.2. Discrimination of different species of sponges from the same genus

-1

Wavenumber/cm

Fig. 3. IR spectra of two polymorphs of calcium carbonate.

Based upon classical spicule identification by Prof. Jinhe Li, a well-recognized expert in sponge identification, AC1 and AC2 are

Please cite this article in press as: J.-H. Gan et al., J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2013.12.088

4

J.-H. Gan et al. / Journal of Molecular Structure xxx (2014) xxx–xxx 1649 3417 1237 1061

1536 1454

2925

Acanthella sp. 1 2854

Absorbance

929

471

801 584 713

1008

1659 3285 2922 1534 1444

2852

1229

Acanthella sp. 2

571 773700

2363

4000.0

3000

2000

1500

464

1000

400.0

Wavenumber/cm-1 Fig. 5. IR spectra of two sponges from Genus Acanthella: Acanthella sp. 1 (AC1) and Acanthella sp. 2 (AC2).

1114 1655

1045

1114 1540

3329

Agelas clathrodes

1045

1411 1443

2924 2852

788

600 475

653 672

788

1082

1646

Absorbance

1517

1082

3301 1452

1226 875 785

2959 2926

Agelas sp. 2

785 712

469

712 700

1082

1468 874 1647 3396

1082

Agelas sp. 3

699 712 712

3000

2000

1500

857

874 857

2928 2521

4000.0

1000

789

472

400.0 1150.0 1000

800 650.0

Wavenumber/cm-1 Fig. 6. IR spectra of three sponges from Genus Agelas: Agelas clathrodes (AG1), Agelas sp. 2 (AG2) and Agelas sp. 3 (AG3).

Aaptos aaptos

Absorbance

1680 1696 1627

1424 1412

1549 1468 1453

855 1323

1242

1112 1080 1043

1514

715 700

875

Plakortis simplex

1660

1549

1468 1403

1278 1235

1081 1017 1043

875 855 829

700

900

700

1453

1696

have similar spectral profile in the range of 1800–1300 cm 1 despiting of small variations. Nevertheless, the strongest peak in AC2 spectra locates at 1008 cm 1 rather than 1649 cm 1. AC2 has a sharp peak at 571 cm 1 and a broad weak peak at 2363 cm 1, which cannot be observed in AC1. Consequently, AC1 and AC2 can be easily distinguished and even their species could be accurately identified if standard specimens could be acquired. Based upon the results of spicule identification, AG1, AG2 and AG3 are considered three different species of Genus Agelas (Class Demospongiae, Order Agelasida, Family Agelasidae) but their exact species cannot be identified as well. AG2 can be easily differentiated from AG1 by comparing the peak profiles in the range of 1300–500 cm 1. The highest peak in the range of AG2 is 1082 cm 1 instead of 1114 cm 1 and other characteristic peaks mainly contributed by CaCO3 (875, 712 and 700 cm 1) can also be observed, which are absent in AG1 (Fig. 6). Surprisingly, the dominant compositions of AG3 are not proteins but mixture of calcite and aragonite as indicated by the strong absorption peaks of CaCO3 (2521, 1787, 1468, 1082, 874, 857, 712 and 699 cm 1). The three specimens can therefore be easily discriminated and we speculate that AG3 is probably a calcareous sponge but not a demosponge.

1512 1638

1800.0 1700 1600 1500 1400 1300 1200 1100 1000

800

600.0

Wavenumber/cm-1 Fig. 7. Second derivative IR spectra of sponges from different orders: Aaptos aaptos (AA) and Plakortis simplex (PL) in the range of 1800–600 cm 1.

two species of Genus Acanthella (Class Demospongiae, Order Halichondrida, Family Axinellidae) but their exact species cannot be identified because of the inherent limitation of spicule identification [4]. Unsurprisingly, like AC1, AC2 also has abundant of proteins indicated by the two strong absorption bands, Amide I (1659 cm 1) and Amide II (1534 cm 1) (Fig. 5). The two species

3.3. Discrimination of sponges with similar IR spectral profile In traditional spicule identification, mistakes are likely to happen when the features of sponge spicules are rather similar [4]. In terms of IR identification, when two sponges have similar IR spectra, second derivative IR spectroscopy (SD-IR) can also be used to amply the tiny differences in FT-IR spectra [5]. Among the nine sponges, AA IR spectrum is highly similar to PL with a correlation coefficient of 0.9488 (Fig. 2) though they are from different orders, so second derivative calculation has been applied to the spectra of AA and PL to afford SD-IR spectra (Fig. 7). Differences between AA and PL can be observed in the range of 1800–600 cm 1. Specifically, the strongest peak in PL is 1638 cm 1 while that in AA is 875 cm 1. In the region of Amide I (1600–1700 cm 1), the three most evident peaks in AA are 1696, 1680, 1627 cm 1 while those in PL are 1696, 1660 and 1638 cm 1, suggesting that they have different proteins/protein profiles. In addition, a group of peaks (1424, 1080, 875, 855, 715 and 700 cm 1) contributed by CaCO3 can be observed, which verifies the results in Section 3.1. To identify the differences between the two sponges more convincingly, 2DCOS-IR synchronous spectra have been applied in the ranges of 900–1300 cm 1 and 1300–1500 cm 1. In synchronous IR spectrum, the auto-peaks on the diagonal line display the self-correlativity and susceptibility of certain absorption band, which produce the variation of spectral intensity through thermal treatment. The cross-peaks at the off-diagonal locations disclose the relativity of intensity variations of a pair of group vibrations corresponding to their frequencies. The closer the correlativity is, the stronger the intensity of cross-peak will be [11]. In 2DCOS-IR spectra, positive correlation (red/green area) indicates that a group of absorption bands change simultaneously (either stronger or weaker), while negative correlation (blue area) is just the reverse. Fig. 8 shows the 2DCOS-IR synchronous spectra of AA and PL in the ranges of 900–1300 cm 1 and 1300–1500 cm 1. Detail information (positions, relative intensities and correlations) of the auto-peaks in Fig. 8 are summarized in Table 2. In the range of 900–1300 cm 1, AA has five strong autopeaks with the strongest peak at 1215 cm 1 while PL has four with 1082 cm 1 the strongest. In the range of 1300–1500 cm 1, AA has three while PL only has one. Form the all above, two sponges have respective unique fingerprints in the range of 900–1500 cm 1 which can be used as an exclusive range for the identification of the two sponges.

Please cite this article in press as: J.-H. Gan et al., J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2013.12.088

5

J.-H. Gan et al. / Journal of Molecular Structure xxx (2014) xxx–xxx

Fig. 8. 2DCOS-IR synchronous spectra of (a) Aaptos aaptos (AA) and (b) Plakortis simplex (PL) in the range of (I) 900–1300 cm

1

and (II) 1300–1500 cm

1

.

Table 2 Strong auto-peaks in 2DCOS-IR synchronous spectra of Aaptos aaptos (AA) and Plakortis simplex (PL). Sponge

Autopeaks/cm

1

(Threshold: 50% of relative intensity)

900–1300

1300–1500

AA

956

987

1060

1215

PL

1043

1082

1243

1279

1248

1452

1461

1467

1456

Notes: Peaks in bold are the strongest auto-peaks in the respective ranges; peaks in underline are in negative correlation with other auto-peaks.

4. Conclusions Nine sponges have been rapidly discriminated by employing Tri-step infrared spectroscopy. Every sponge has unique IR macro-fingerprint. Sponges from different classes and different orders have been evidently discriminated by FT-IR and SD-IR. More importantly, the sponges from the same genus which could not be identified by traditional spicule identification have been definitely distinguished and even their species could be accurately identified if standard specimens could be acquired. For sponges having highly similar chemical profile (IR spectral profile), SD-IR and 2DCOS-IR have been applied to enhance the spectral resolution to distinguish AA and PL convincingly. It has been demonstrated that the Tri-step infrared spectroscopy could be a promising tool for sponge identification in a more objective, simpler and timesaving manner. Acknowledgements This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (81225023), the

National Natural Science Fund of China (Nos. 81072573, 81172978, 41106127, and 81001394), and Shanghai Subject Chief Scientist (12XD1400200). References [1] W. Bergmann, D.C. Burke, J. Org. Chem. 20 (11) (1955) 1501–1507. [2] J.W. Blunt, B.R. Copp, W.-P. Hu, M.H.G. Munro, P.T. Northcote, M.R. Prinsep, Nat. Product Rep. 26 (2) (2009) 170–244. [3] J.B. McClintock, B.J. Baker, Marine Chemical Ecology, first ed., CRC Press, 2001. [4] J.N.A. Hooper, R.W.M.v. Soest, P. Willenz, Systema Porifera. A Guide to the Classification of Sponges, vol. 1 & 2, Springer, US: New York, 2002. p. 1708. [5] S.Q. Sun, J.B. Chen, Q. Zhou, G.H. Lu, K. Chan, Planta Medica 76 (2010) 1987– 1996. [6] S.Q. Sun, Q. Zhou, J.B. Chen, Infrared Spectroscopy for Complex Mixtures, Chemical Industry Press, Beijing, 2011. [7] C. Xu, X. Jia, R. Xu, Y. Wang, Q. Zhou, S. Sun, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 114 (2013) 421–431. [8] Y. Wang, C.-H. Xu, P. Wang, S.-Q. Sun, J.-B. Chen, J. Li, T. Chen, J.-B. Wang, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 83 (1) (2011) 265–270. [9] C. Xu, Y. Wang, J. Chen, Q. Zhou, P. Wang, Y. Yang, S. Sun, J. Pharm. Biomed. Anal. 74 (2013) 298–307. [10] J. Kong, S. Yu, Acta Biochimica et Biophysica Sinica 39 (8) (2007) 549–559. [11] I. Noda, J. Am. Chem. Soc. 111 (21) (1989) 8116–8118.

Please cite this article in press as: J.-H. Gan et al., J. Mol. Struct. (2014), http://dx.doi.org/10.1016/j.molstruc.2013.12.088