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Analysis and discrimination of ten different sponges by multi-step infrared spectroscopy
Chinese Chemical Letters 26 (2015) 215–220
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Analysis and discrimination of ten different sponges by multi-step infrared spectroscopy Jian-Hong Gan a,e,1, Chang-Hua Xu a,1, Hong-Zhe Zhu d, Fang Mao a, Fan Yang b,*, Qun Zhou c, Su-Qin Sun c,* a
College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China Key Laboratory for Marine Drugs, Department of Pharmacy, State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China c Department of Chemistry, Tsinghua University, Beijing 100084, China d Department of Pharmacy, Taian Maternal and Child Health Hospital, Jiangsu 271000, China e Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2014 Received in revised form 10 December 2014 Accepted 22 December 2014 Available online 21 January 2015
In this study, a convenient method using multi-step infrared spectroscopy, including Fourier transform infrared spectroscopy (FT-IR), second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR), was employed to analyze and discriminate ten marine sponges from two classes collected from the Xisha Islands in the South China Sea. Each sponge had an exclusive macroscopic fingerprint. From the IR spectra, it was noted that the main ingredient of calcareous sponges was calcium carbonate, but that of demosponges was proteins. For sponges from the same genus or having highly similar chemical profile (IR spectral profile), SD-IR and 2DCOS-IR were applied to successfully reveal the tiny differences. It was demonstrated that the multi-step infrared spectroscopy was a feasible and objective approach for marine sponge identification. ß 2015 Fan Yang and Su-Qin Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Keywords: Sponge Discrimination Infrared spectroscopy Second derivative infrared spectroscopy Two-dimensional correlation infrared spectroscopy
1. Introduction Marine sponges are classified into three classes: Calcarea, Hexactinellida and Demospongiae containing 25 orders, 127 families and 790 genera. There are approximately 15,000 species reported worldwide [1–4]. Almost 1/3 marine natural products come from sponges [3,5]. In all the prevalent traditional ways to identify sponges, the most accurate and commonly used method is spicule identification, which examines sponge skeletal structure. This method routinely requires a whole piece of sponge and heavily depends on experts mastering with English, French and German and ages of experience [6]. Now the number of experts in the art of sponge identification is greatly reduced. Hence, it is necessary to establish more objective and simpler methods to discriminate marine sponges.
‘‘Multi-step infrared spectroscopy’’ including FT-IR, SD-IR and 2DCOS-IR has the above advantages for sponge identification. FT-IR has been proved to be a quick, simple and effective method with good signal-to-noise ratio and excellent repeatability to investigate complicated mixtures such as herbal medicine (HM) [7,8]. To delineate the overlapped spectra in FT-IR, second derivative infrared spectroscopy (SD-IR) is used to improve the apparent resolution [9]. If the differences are still too small to discriminate, two-dimensional correlation infrared spectroscopy (2DCOS-IR) can be adopted to illustrate FT-IR spectra in a second dimension to reveal the differences more convincingly [10,11]. Ten different sponges collected from Xisha Islands in South China Sea of two classes, five orders and ten species have been studied using the multi-step infrared spectroscopy to establish a new method to discriminate sponges in a more objective, direct and quicker manner. 2. Experimental
* Corresponding authors. E-mail addresses: [email protected] (F. Yang), [email protected] (S.-Q. Sun). 1 Author contributions: Jian-hong Gan and Chang-hua Xu had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. These authors contributed equally to this study.
2.1. Apparatus A spectrum GX FT-IR spectrometer (PerkinElmer, UK) equipped with a DTGS detector and a scanning range from 400 to 4000 cm1
http://dx.doi.org/10.1016/j.cclet.2015.01.012 1001-8417/ß 2015 Fan Yang and Su-Qin Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
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Table 1 Ten China sponges collected from Xisha Islands of South China Sea. Class
Order
Family
Genus
Specie
Abbreviation
Calcarea
Leucosolenida
Grantiidae
Leucandra
sp
LA
Leucosolenidae
Leuconia
sp
LO
Poecilosclerida
Mycalidae
Mycale
Fibrexilis
MY
Dietyoceratida
Thorectidae
Hyrtios
sp
HY
Spongiidae
Hippospongia
Lachna
HI
Aplysinopsis
sp
AP
Halichondridae
Halichondria
sp
HA
Axinelliedae
Phakellia
Fusca Thiele
PA
Acanthella
sp1
AC1
sp2
AC2
Demospongiae
Halichondrida
Photo
with a 4 cm1 resolution was used. Spectra were calculated from a total of 32 scans at 0.2 cm/s of OPD speed. The interferences of H2O and CO2 were eliminated during scanning. A CKW-II programmable temperature controller (Beijing Chaoyang Automatic Instrument Co., China) was employed to perform the thermal perturbation from the range 50 8C to 120 8C. Spectra were collected at 10 8C intervals. The second derivative IR spectra were gained after 13-point smoothing of the IR spectra by Savitzky–Golay polynomial fitting. Two-dimensional IR correlation spectra were obtained after using 2D correlation analysis software (developed by IR Lab, Tsinghua University) to analyze the series of thermo perturbation dynamic spectra.
3. Results and discussion
2.2. Samples and reagents
3.1. Analysis and discrimination of five sponges from two classes
Sponges (Table 1) were obtained from Xisha Islands in South China Sea in April 2007 and 2009 (kept in Department of Pharmacy, Changzheng Hospital, Second Military Medical University) and
Fig. 1 shows the IR spectra of five sponges belonging to two classes. In these five sponges, Leucandra sp. is a calcareous sponge and is collected for the first time in China. The main composition of
were authenticated by Prof. Jinhe Li, Qingdao Institute of Oceanology, Chinese Academy of Sciences. KBr was bought from Sigma (St. Louis, MO, USA). 2.3. Procedure Sponge samples were desiccated in vacuo and then were grounded into powder before IR measurement. Each sponge sample (about 1–2 mg) was mixed with KBr (100 mg), grounded into powder (200 mesh), and then pressed into a tablet.
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
217
Fig. 1. IR spectra of five sponges from different classes and orders: Aplysinopsis sp. (AP), Hyrtios sp. (HY), Hippospongia lachna (HI), Mycale fibrexilis (MY) and Leucandra sp. (LA).
Leucandra sp. is CaCO3 with five peaks at 2523, 1803, 1418, 874, 715 cm1. The other four sponges are demosponges and their main compositions are proteins with two characteristic absorption bands peaks: Amide I (1650 cm1) and amide II (1535 cm1). The attribution of main absorption peaks of the five sponges is summarized in Table 2. The IR spectra of the four demosponges, from three orders, four families, are different according to the group-peak matching technique [12,13]. The peaks at 1422, 875, 712 cm1 and 1082, 855, 712, 699 cm1 of AP suggest that it contains calcium carbonate of both calcite and aragonite forms. The peaks at 873 cm1 and 2522, 1468, 1082, 855, 712, 699 cm1 of HI indicate
that HI contains more aragonite than calcite. HY (1080, 873, 855, 712, 699 cm1) and MY (878, 707 cm1) contains both forms of CaCO3 but with lower contents. Thus, the four demosponges from different five orders can be distinguished readily (Fig. 2). By spicule identification, LA and LO belong to the same order (class Calcarea, order Leucosolenida), but their exact species cannot be identified. CaCO3 is the main component of the two sponges. LA can be easily differentiated from LO through comparing the peak profiles in the range of 2000–1000 cm1 (Figs. 3 and 4). The peaks at 1514 and 1043 cm1 of LA are evidently higher than those of LO and the peak at 1412 cm1 of LO is stronger than that of LA. Hence, the two specimens can be easily discriminated.
Table 2 Preliminary assignment of main peaks in FT-IR spectra of five sponges. Band (cm1) and main attribution
Sponge Aplysinopsis sp. (AP)
Hyrtios sp. (HY)
Hippospongia lachna (HI)
Mycale fibrexilis (MY)
Leucandra sp. (LA)
Proteins
1600–1690 (Amide I) 1480–1575 (Amide II) [9]
1656 1528
1649 1535
1655 1515
1648 1530
1647
Calcite
2515 1800 1420 875 713
Composition
Aragonite
2522 1788 1473 1080 855 713 700
1422 875 712
873
873
878
2523 1803 1418 874 715
2522
1082 855 712 699
1082 856 712 699
1468 1082 855 712 699
3302
3311
3389
2960
2959
2926
2924, 2853
Compounds with OH or/and NH groups
3500–3200 (n(OH, NH))
3300
Compounds with CH2 or/and CH3 groups
2961 (n (CH3)) 2929, 2854 (n (CH2)) 1440–1555 (d(CH2, CH3))
2961 2929
2926
1450
1451
1083 707
1438
3395
2930
218
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
Fig. 2. SD-IR spectra of five sponges from different classes and orders: Aplysinopsis sp. (AP), Hyrtios sp. (HY), Hippospongia lachna (HI), Mycale fibrexilis (MY) and Leucandra sp. (LA).
Fig. 3. IR spectra of two calcareous sponges from the same order Leucosolenida: Leucandra sp. (LA) and Leuconia sp. (LO).
Fig. 4. SD-IR spectra of two calcareous sponges from the same order Leucosolenida: Leucandra sp. (LA) and Leuconia sp. (LO).
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
219
Fig. 5. IR spectra of four sponges from the same order Halichondrida: Halichondri sp. (HA), Phakellia fusca (PH), Acanthella sp1 (AC1) and Acanthella sp2 (AC2).
3.2. Discrimination of different species from the same genus, different genus from the same family and different families of sponges from the same order HA belongs to the same order with PA, AC1 and AC2. PA is in the same family with AC1 and AC2. AC1 and AC2 are in the same genus (Table 1). The exact species of AC1 and AC2 cannot be discriminated for the inherent limitations of spicule identification [14,15]. Two strong absorption bands, amide I (1649 cm1) and amide II (1536 cm1) (Fig. 5), suggest the existence of abundant proteins. The peaks at 1113 and 874 cm1 of HA clearly differ from those of PH, AC1 and AC2. The peak at 1081 cm1 of PH is stronger than that of AC1 and AC2. AC1 and AC2 have similar spectral profile in the range of 1800–1300 cm1 with small variations: a sharp peak at 1238 cm1 in AC1 can be seen clearly other than that in
AC2. Thus, these four sponges can be distinguished and even their species can be accurately identified if standard specimens are available. Mistakes can occur in traditional spicule identification when the characteristics of sponge spicules are similar [14]. With regards to IR identification, if two sponges have close IR spectra, second derivative IR spectroscopy (SD-IR) with higher resolution can be applied to show the minor differences in FT-IR spectra [9]. The IR spectra of AC1 and AC2 are similar except for the absorption band around 1238 cm1. In order to discriminate the two sponges more clearly, SD-IR and 2DCOS-IR are used to analyze AC1 and AC2 [15]. In SD-IR spectra, overlapped peaks can be divided into two or more peaks and thus hidden peaks can be observed. Specifically, two peaks at 1236 and 875 cm1 of AC1 are stronger than those of AC2 in SD-IR (Fig. 6).
Fig. 6. Second derivative IR spectra of four sponges from the same order Halichondrida: Halichondri sp. (HA), Phakellia fusca (PH), Acanthella sp1 (AC1) and Acanthella sp2 (AC2) in the range of 1800–600 cm1.
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
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Fig. 7. 2DCOS-IR synchronous spectra of (a) Acanthella sp1 (AC1) and (b) Acanthella sp2 (AC2) in the range of 900–1300 cm1 (I) and 1300–1500 cm1 (II).
Table 3 Strong auto-peaks in 2DCOS-IR synchronous spectra of Acanthella sp1 (AC1) and (b) Acanthella sp2 (AC2). Sponge
Autopeaks/cm1 (threshold: 50% of relative intensity) 1300–1500
900–1300 AC1 AC2
931 980
1035
1084 1087
1110 1188
1217
1244
1458 1456
1469 1471
Notes: Peaks in bold are the strongest auto-peaks in the respective ranges.
A more accurate approach, 2DCOS-IR synchronous spectroscopy is used to clearly differentiate the two sponges in the ranges of 900–1300 cm1 and 1300–1500 cm1. The auto-peaks on the diagonal line in synchronous IR spectrum show the susceptibility and self-correlativity of certain absorption bands, which cause the change of spectral intensity with thermal treatment. While the cross-peaks at the off-diagonal locations display 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 [16]. Positive correlation (red/green area) in 2DCOS-IR spectra indicates that a group of absorption bands change simultaneously (either stronger or weaker), while negative correlation (blue area) suggests the opposite. For easy comparison, detail information (positions, relative intensities and correlations) of the autopeaks of AC1 and AC2 in Fig. 7 is summarized in Table 3. Six strong autopeaks with the strongest at 980 cm1 can be observed in the 2DCOS-IR spectrum of AC2 in the range of 900–1300 cm1 while only two peaks with 1110 cm1 being the strongest can be seen in AC1 spectrum. However, AC1 and AC2 both have two strong autopeaks despite of small differences in the range of 1300–1500 cm1. Based on above observations, the unique fingerprints of AC1 and AC2 in the range of 900–1300 cm1 can be used as exclusive features to discriminate the two sponges. 4. Conclusion The multi-step infrared spectroscopy allowed ten sponges to be discriminated rapidly. Sponges from different genus, different classes and different orders have their unique IR macro-fingerprints. If the IR spectra of standard specimens could be acquired, even the exact species of sponges could be accurately identified. Meanwhile, SD-IR and 2DCOS-IR have been applied to successfully distinguish the subtle differences of sponges with similar chemical profiles. It has been demonstrated that the multi-step infrared spectroscopy can be a promising objective method for rapid and accurate identification of marine sponges.
Acknowledgments This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (No. 81225023), the National Natural Science Fund of China (Nos. 41476121, 81302691, 81172978), the Innovation Program of Shanghai Municipal Education Commission (No. 14YZ037) and partially supported by Shanghai Subject Chief Scientist (No. 12XD1400200). We are also grateful for the financial support of the National High Technology Research and Development Program of China (863 Projects, No. 2013AA092902). References [1] J.N.A. Hooper, Coral reef sponges of the Sahul shelf – a case for habitat preservation, Mem. Qld. Mus. 36 (1994) 93–106. [2] I. Hermawan, N.J. de Voogd, J. Tanaka, An acetylenic alkaloid from the calcareous sponge Leucetta sp., Mar. Drugs 9 (2011) 382–386. [3] J.W. Blunt, B.R. Copp, W.P. Hu, et al., Marine natural products, Natl. Prod. Rep. 26 (2009) 170–244. [4] J.B. McClintock, B.J. Baker, Marine Chemical Ecology, CRC Press, New York, 2001. [5] H.X. Ding, L.C. Da, R.C. Yang, et al., First total synthesis of a naturally occurring nucleoside disulfide: 9-(50 -deoxy-50 -thio-b-D-xylofuranosyl)adenine disulfide, Chin. Chem. Lett. 23 (2012) 996–998. [6] P. Botting, L.A. Muir, S.H. Xiao, X.F. Li, J.P. Lin, Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China, Lethaia 45 (2012) 463–475. [7] S.Q. Sun, Q. Zhou, J.B. Chen, Infrared Spectroscopy for Complex Mixtures: Applications in Food and Traditional Chinese Medicine, Chemical Industry Press, Beijing, 2011. [8] H.Y. Fu, D.C. Huang, T.M. Yang, Y.B. She, H. Zhang, Rapid recognition of Chinese herbal pieces of Areca catechu by different concocted processes using Fourier transform mid-infrared spectroscopy and near-infrared spectroscopy combined with partial least-squares discriminant analysis, Chin. Chem. Lett. 24 (2013) 639–642. [9] S.Q. Sun, J.B. Chen, Q. Zhou, G.H. Lu, K. Chan, Application of mid-infrared spectroscopy in the quality control of traditional Chinese medicines, Planta Med. 76 (2010) 1987–1996. [10] C.H. Xu, X.G. Jia, R. Xu, et al., Rapid discrimination of Herba Cistanches by multi-step infrared macro-fingerprinting combined with soft independent modeling of class analogy (SIMCA), Spectrochim. Acta A 114 (2013) 421–431. [11] Y. Wang, C.H. Xu, P. Wang, et al., Analysis and identification of different animal horns by a three-stage infrared spectroscopy, Spectrochim. Acta A 83 (2011) 265– 270. [12] C.H. Xu, Y. Wang, J.B. Chen, et al., Infrared macro-fingerprint analysis-throughseparation for holographic chemical characterization of herbal medicine, J. Pharm. Biomed. Anal. 74 (2013) 298–307. [13] J. Kong, S.N. Yu, Fourier transform infrared spectroscopic analysis of protein secondary structures, Acta Biochim. Biophys. Sin. 39 (2007) 549–559. [14] J.N.A. Hooper, R.W.M. Van Soest, P. Willenz, Systema Porifera. A Guide to the Classification of Sponges, Springer-Verlag, New York, 2002. [15] J.H. Gan, C.H. Xu, H.B. Yu, et al., Rapid discrimination of china sponges by tri-step infrared spectroscopy: a prelimiary study, J. Mol. Struct. 1069 (2014) 147–151. [16] I. Noda, Two-dimensional infrared spectroscopy, J. Am. Chem. Soc. 111 (1989) 8116–8118.
Contents lists available at ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Analysis and discrimination of ten different sponges by multi-step infrared spectroscopy Jian-Hong Gan a,e,1, Chang-Hua Xu a,1, Hong-Zhe Zhu d, Fang Mao a, Fan Yang b,*, Qun Zhou c, Su-Qin Sun c,* a
College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China Key Laboratory for Marine Drugs, Department of Pharmacy, State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China c Department of Chemistry, Tsinghua University, Beijing 100084, China d Department of Pharmacy, Taian Maternal and Child Health Hospital, Jiangsu 271000, China e Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200433, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2014 Received in revised form 10 December 2014 Accepted 22 December 2014 Available online 21 January 2015
In this study, a convenient method using multi-step infrared spectroscopy, including Fourier transform infrared spectroscopy (FT-IR), second derivative infrared spectroscopy (SD-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR), was employed to analyze and discriminate ten marine sponges from two classes collected from the Xisha Islands in the South China Sea. Each sponge had an exclusive macroscopic fingerprint. From the IR spectra, it was noted that the main ingredient of calcareous sponges was calcium carbonate, but that of demosponges was proteins. For sponges from the same genus or having highly similar chemical profile (IR spectral profile), SD-IR and 2DCOS-IR were applied to successfully reveal the tiny differences. It was demonstrated that the multi-step infrared spectroscopy was a feasible and objective approach for marine sponge identification. ß 2015 Fan Yang and Su-Qin Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
Keywords: Sponge Discrimination Infrared spectroscopy Second derivative infrared spectroscopy Two-dimensional correlation infrared spectroscopy
1. Introduction Marine sponges are classified into three classes: Calcarea, Hexactinellida and Demospongiae containing 25 orders, 127 families and 790 genera. There are approximately 15,000 species reported worldwide [1–4]. Almost 1/3 marine natural products come from sponges [3,5]. In all the prevalent traditional ways to identify sponges, the most accurate and commonly used method is spicule identification, which examines sponge skeletal structure. This method routinely requires a whole piece of sponge and heavily depends on experts mastering with English, French and German and ages of experience [6]. Now the number of experts in the art of sponge identification is greatly reduced. Hence, it is necessary to establish more objective and simpler methods to discriminate marine sponges.
‘‘Multi-step infrared spectroscopy’’ including FT-IR, SD-IR and 2DCOS-IR has the above advantages for sponge identification. FT-IR has been proved to be a quick, simple and effective method with good signal-to-noise ratio and excellent repeatability to investigate complicated mixtures such as herbal medicine (HM) [7,8]. To delineate the overlapped spectra in FT-IR, second derivative infrared spectroscopy (SD-IR) is used to improve the apparent resolution [9]. If the differences are still too small to discriminate, two-dimensional correlation infrared spectroscopy (2DCOS-IR) can be adopted to illustrate FT-IR spectra in a second dimension to reveal the differences more convincingly [10,11]. Ten different sponges collected from Xisha Islands in South China Sea of two classes, five orders and ten species have been studied using the multi-step infrared spectroscopy to establish a new method to discriminate sponges in a more objective, direct and quicker manner. 2. Experimental
* Corresponding authors. E-mail addresses: [email protected] (F. Yang), [email protected] (S.-Q. Sun). 1 Author contributions: Jian-hong Gan and Chang-hua Xu had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. These authors contributed equally to this study.
2.1. Apparatus A spectrum GX FT-IR spectrometer (PerkinElmer, UK) equipped with a DTGS detector and a scanning range from 400 to 4000 cm1
http://dx.doi.org/10.1016/j.cclet.2015.01.012 1001-8417/ß 2015 Fan Yang and Su-Qin Sun. Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. All rights reserved.
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J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
Table 1 Ten China sponges collected from Xisha Islands of South China Sea. Class
Order
Family
Genus
Specie
Abbreviation
Calcarea
Leucosolenida
Grantiidae
Leucandra
sp
LA
Leucosolenidae
Leuconia
sp
LO
Poecilosclerida
Mycalidae
Mycale
Fibrexilis
MY
Dietyoceratida
Thorectidae
Hyrtios
sp
HY
Spongiidae
Hippospongia
Lachna
HI
Aplysinopsis
sp
AP
Halichondridae
Halichondria
sp
HA
Axinelliedae
Phakellia
Fusca Thiele
PA
Acanthella
sp1
AC1
sp2
AC2
Demospongiae
Halichondrida
Photo
with a 4 cm1 resolution was used. Spectra were calculated from a total of 32 scans at 0.2 cm/s of OPD speed. The interferences of H2O and CO2 were eliminated during scanning. A CKW-II programmable temperature controller (Beijing Chaoyang Automatic Instrument Co., China) was employed to perform the thermal perturbation from the range 50 8C to 120 8C. Spectra were collected at 10 8C intervals. The second derivative IR spectra were gained after 13-point smoothing of the IR spectra by Savitzky–Golay polynomial fitting. Two-dimensional IR correlation spectra were obtained after using 2D correlation analysis software (developed by IR Lab, Tsinghua University) to analyze the series of thermo perturbation dynamic spectra.
3. Results and discussion
2.2. Samples and reagents
3.1. Analysis and discrimination of five sponges from two classes
Sponges (Table 1) were obtained from Xisha Islands in South China Sea in April 2007 and 2009 (kept in Department of Pharmacy, Changzheng Hospital, Second Military Medical University) and
Fig. 1 shows the IR spectra of five sponges belonging to two classes. In these five sponges, Leucandra sp. is a calcareous sponge and is collected for the first time in China. The main composition of
were authenticated by Prof. Jinhe Li, Qingdao Institute of Oceanology, Chinese Academy of Sciences. KBr was bought from Sigma (St. Louis, MO, USA). 2.3. Procedure Sponge samples were desiccated in vacuo and then were grounded into powder before IR measurement. Each sponge sample (about 1–2 mg) was mixed with KBr (100 mg), grounded into powder (200 mesh), and then pressed into a tablet.
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
217
Fig. 1. IR spectra of five sponges from different classes and orders: Aplysinopsis sp. (AP), Hyrtios sp. (HY), Hippospongia lachna (HI), Mycale fibrexilis (MY) and Leucandra sp. (LA).
Leucandra sp. is CaCO3 with five peaks at 2523, 1803, 1418, 874, 715 cm1. The other four sponges are demosponges and their main compositions are proteins with two characteristic absorption bands peaks: Amide I (1650 cm1) and amide II (1535 cm1). The attribution of main absorption peaks of the five sponges is summarized in Table 2. The IR spectra of the four demosponges, from three orders, four families, are different according to the group-peak matching technique [12,13]. The peaks at 1422, 875, 712 cm1 and 1082, 855, 712, 699 cm1 of AP suggest that it contains calcium carbonate of both calcite and aragonite forms. The peaks at 873 cm1 and 2522, 1468, 1082, 855, 712, 699 cm1 of HI indicate
that HI contains more aragonite than calcite. HY (1080, 873, 855, 712, 699 cm1) and MY (878, 707 cm1) contains both forms of CaCO3 but with lower contents. Thus, the four demosponges from different five orders can be distinguished readily (Fig. 2). By spicule identification, LA and LO belong to the same order (class Calcarea, order Leucosolenida), but their exact species cannot be identified. CaCO3 is the main component of the two sponges. LA can be easily differentiated from LO through comparing the peak profiles in the range of 2000–1000 cm1 (Figs. 3 and 4). The peaks at 1514 and 1043 cm1 of LA are evidently higher than those of LO and the peak at 1412 cm1 of LO is stronger than that of LA. Hence, the two specimens can be easily discriminated.
Table 2 Preliminary assignment of main peaks in FT-IR spectra of five sponges. Band (cm1) and main attribution
Sponge Aplysinopsis sp. (AP)
Hyrtios sp. (HY)
Hippospongia lachna (HI)
Mycale fibrexilis (MY)
Leucandra sp. (LA)
Proteins
1600–1690 (Amide I) 1480–1575 (Amide II) [9]
1656 1528
1649 1535
1655 1515
1648 1530
1647
Calcite
2515 1800 1420 875 713
Composition
Aragonite
2522 1788 1473 1080 855 713 700
1422 875 712
873
873
878
2523 1803 1418 874 715
2522
1082 855 712 699
1082 856 712 699
1468 1082 855 712 699
3302
3311
3389
2960
2959
2926
2924, 2853
Compounds with OH or/and NH groups
3500–3200 (n(OH, NH))
3300
Compounds with CH2 or/and CH3 groups
2961 (n (CH3)) 2929, 2854 (n (CH2)) 1440–1555 (d(CH2, CH3))
2961 2929
2926
1450
1451
1083 707
1438
3395
2930
218
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
Fig. 2. SD-IR spectra of five sponges from different classes and orders: Aplysinopsis sp. (AP), Hyrtios sp. (HY), Hippospongia lachna (HI), Mycale fibrexilis (MY) and Leucandra sp. (LA).
Fig. 3. IR spectra of two calcareous sponges from the same order Leucosolenida: Leucandra sp. (LA) and Leuconia sp. (LO).
Fig. 4. SD-IR spectra of two calcareous sponges from the same order Leucosolenida: Leucandra sp. (LA) and Leuconia sp. (LO).
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
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Fig. 5. IR spectra of four sponges from the same order Halichondrida: Halichondri sp. (HA), Phakellia fusca (PH), Acanthella sp1 (AC1) and Acanthella sp2 (AC2).
3.2. Discrimination of different species from the same genus, different genus from the same family and different families of sponges from the same order HA belongs to the same order with PA, AC1 and AC2. PA is in the same family with AC1 and AC2. AC1 and AC2 are in the same genus (Table 1). The exact species of AC1 and AC2 cannot be discriminated for the inherent limitations of spicule identification [14,15]. Two strong absorption bands, amide I (1649 cm1) and amide II (1536 cm1) (Fig. 5), suggest the existence of abundant proteins. The peaks at 1113 and 874 cm1 of HA clearly differ from those of PH, AC1 and AC2. The peak at 1081 cm1 of PH is stronger than that of AC1 and AC2. AC1 and AC2 have similar spectral profile in the range of 1800–1300 cm1 with small variations: a sharp peak at 1238 cm1 in AC1 can be seen clearly other than that in
AC2. Thus, these four sponges can be distinguished and even their species can be accurately identified if standard specimens are available. Mistakes can occur in traditional spicule identification when the characteristics of sponge spicules are similar [14]. With regards to IR identification, if two sponges have close IR spectra, second derivative IR spectroscopy (SD-IR) with higher resolution can be applied to show the minor differences in FT-IR spectra [9]. The IR spectra of AC1 and AC2 are similar except for the absorption band around 1238 cm1. In order to discriminate the two sponges more clearly, SD-IR and 2DCOS-IR are used to analyze AC1 and AC2 [15]. In SD-IR spectra, overlapped peaks can be divided into two or more peaks and thus hidden peaks can be observed. Specifically, two peaks at 1236 and 875 cm1 of AC1 are stronger than those of AC2 in SD-IR (Fig. 6).
Fig. 6. Second derivative IR spectra of four sponges from the same order Halichondrida: Halichondri sp. (HA), Phakellia fusca (PH), Acanthella sp1 (AC1) and Acanthella sp2 (AC2) in the range of 1800–600 cm1.
J.-H. Gan et al. / Chinese Chemical Letters 26 (2015) 215–220
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Fig. 7. 2DCOS-IR synchronous spectra of (a) Acanthella sp1 (AC1) and (b) Acanthella sp2 (AC2) in the range of 900–1300 cm1 (I) and 1300–1500 cm1 (II).
Table 3 Strong auto-peaks in 2DCOS-IR synchronous spectra of Acanthella sp1 (AC1) and (b) Acanthella sp2 (AC2). Sponge
Autopeaks/cm1 (threshold: 50% of relative intensity) 1300–1500
900–1300 AC1 AC2
931 980
1035
1084 1087
1110 1188
1217
1244
1458 1456
1469 1471
Notes: Peaks in bold are the strongest auto-peaks in the respective ranges.
A more accurate approach, 2DCOS-IR synchronous spectroscopy is used to clearly differentiate the two sponges in the ranges of 900–1300 cm1 and 1300–1500 cm1. The auto-peaks on the diagonal line in synchronous IR spectrum show the susceptibility and self-correlativity of certain absorption bands, which cause the change of spectral intensity with thermal treatment. While the cross-peaks at the off-diagonal locations display 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 [16]. Positive correlation (red/green area) in 2DCOS-IR spectra indicates that a group of absorption bands change simultaneously (either stronger or weaker), while negative correlation (blue area) suggests the opposite. For easy comparison, detail information (positions, relative intensities and correlations) of the autopeaks of AC1 and AC2 in Fig. 7 is summarized in Table 3. Six strong autopeaks with the strongest at 980 cm1 can be observed in the 2DCOS-IR spectrum of AC2 in the range of 900–1300 cm1 while only two peaks with 1110 cm1 being the strongest can be seen in AC1 spectrum. However, AC1 and AC2 both have two strong autopeaks despite of small differences in the range of 1300–1500 cm1. Based on above observations, the unique fingerprints of AC1 and AC2 in the range of 900–1300 cm1 can be used as exclusive features to discriminate the two sponges. 4. Conclusion The multi-step infrared spectroscopy allowed ten sponges to be discriminated rapidly. Sponges from different genus, different classes and different orders have their unique IR macro-fingerprints. If the IR spectra of standard specimens could be acquired, even the exact species of sponges could be accurately identified. Meanwhile, SD-IR and 2DCOS-IR have been applied to successfully distinguish the subtle differences of sponges with similar chemical profiles. It has been demonstrated that the multi-step infrared spectroscopy can be a promising objective method for rapid and accurate identification of marine sponges.
Acknowledgments This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (No. 81225023), the National Natural Science Fund of China (Nos. 41476121, 81302691, 81172978), the Innovation Program of Shanghai Municipal Education Commission (No. 14YZ037) and partially supported by Shanghai Subject Chief Scientist (No. 12XD1400200). We are also grateful for the financial support of the National High Technology Research and Development Program of China (863 Projects, No. 2013AA092902). References [1] J.N.A. Hooper, Coral reef sponges of the Sahul shelf – a case for habitat preservation, Mem. Qld. Mus. 36 (1994) 93–106. [2] I. Hermawan, N.J. de Voogd, J. Tanaka, An acetylenic alkaloid from the calcareous sponge Leucetta sp., Mar. Drugs 9 (2011) 382–386. [3] J.W. Blunt, B.R. Copp, W.P. Hu, et al., Marine natural products, Natl. Prod. Rep. 26 (2009) 170–244. [4] J.B. McClintock, B.J. Baker, Marine Chemical Ecology, CRC Press, New York, 2001. [5] H.X. Ding, L.C. Da, R.C. Yang, et al., First total synthesis of a naturally occurring nucleoside disulfide: 9-(50 -deoxy-50 -thio-b-D-xylofuranosyl)adenine disulfide, Chin. Chem. Lett. 23 (2012) 996–998. [6] P. Botting, L.A. Muir, S.H. Xiao, X.F. Li, J.P. Lin, Evidence for spicule homology in calcareous and siliceous sponges: biminerallic spicules in Lenica sp. from the Early Cambrian of South China, Lethaia 45 (2012) 463–475. [7] S.Q. Sun, Q. Zhou, J.B. Chen, Infrared Spectroscopy for Complex Mixtures: Applications in Food and Traditional Chinese Medicine, Chemical Industry Press, Beijing, 2011. [8] H.Y. Fu, D.C. Huang, T.M. Yang, Y.B. She, H. Zhang, Rapid recognition of Chinese herbal pieces of Areca catechu by different concocted processes using Fourier transform mid-infrared spectroscopy and near-infrared spectroscopy combined with partial least-squares discriminant analysis, Chin. Chem. Lett. 24 (2013) 639–642. [9] S.Q. Sun, J.B. Chen, Q. Zhou, G.H. Lu, K. Chan, Application of mid-infrared spectroscopy in the quality control of traditional Chinese medicines, Planta Med. 76 (2010) 1987–1996. [10] C.H. Xu, X.G. Jia, R. Xu, et al., Rapid discrimination of Herba Cistanches by multi-step infrared macro-fingerprinting combined with soft independent modeling of class analogy (SIMCA), Spectrochim. Acta A 114 (2013) 421–431. [11] Y. Wang, C.H. Xu, P. Wang, et al., Analysis and identification of different animal horns by a three-stage infrared spectroscopy, Spectrochim. Acta A 83 (2011) 265– 270. [12] C.H. Xu, Y. Wang, J.B. Chen, et al., Infrared macro-fingerprint analysis-throughseparation for holographic chemical characterization of herbal medicine, J. Pharm. Biomed. Anal. 74 (2013) 298–307. [13] J. Kong, S.N. Yu, Fourier transform infrared spectroscopic analysis of protein secondary structures, Acta Biochim. Biophys. Sin. 39 (2007) 549–559. [14] J.N.A. Hooper, R.W.M. Van Soest, P. Willenz, Systema Porifera. A Guide to the Classification of Sponges, Springer-Verlag, New York, 2002. [15] J.H. Gan, C.H. Xu, H.B. Yu, et al., Rapid discrimination of china sponges by tri-step infrared spectroscopy: a prelimiary study, J. Mol. Struct. 1069 (2014) 147–151. [16] I. Noda, Two-dimensional infrared spectroscopy, J. Am. Chem. Soc. 111 (1989) 8116–8118.