MOLECULAR STRUCTURE AND SPECTRA OF DANTHRON AND EMODIN STUDIED BY FTIR, RAMAN SPECTROSCOPY AND DFT TECHNIQUES

Received 3 August 2020; revised 16 November 2020; accepted 24 November 2020

1. Introduction

Since ancient times anthraquinone derivatives (anthraquinones) obtained from different plant sources have been extensively used as medicines because of their laxative property. Recent studies confirm that these molecules also have anti-inflammatory, analgesic, antimicrobial and an-titumor activities  [1]. Many traditional Chinese medicines (TCMs) are anthraquinone derivatives in nature. Because of the  diversity in their functions these medicines are becoming important sources for potential therapeutic agents. But these medicines generally contain thousands of components. So it becomes very essential to analyse them and find out the exact components that bring out different biological functions. According to their therapeutic potentials different methods have been used to explore the phytochemical and pharmacological effects of TCMs [2]. Reports suggest that the methanolic extract of rhubarb (traditional Chinese medicine) has phenolic compounds in high concentrations, and also exhibits free radical scavenging actions and shows strong antioxidant properties  [3, 4]. The  dihydroxy anthraquinones are the major biologically active molecules in rhubarb  [4, 5]. One of the  dihydroxy anthraquinone derivatives, which is naturally found in barks and roots of many plants and also in Chinese herbs like Rheum officinale and Polygonam cuspidatum, is emodin that has many therapeutic applications [6]. Many studies have reported about the  biological actions of dihydroxy anthraquinones like dan-thron, physcion, aloe-emodin and chrysophanol. Though there are many investigations done for the functional aspect of these dihydroxy derivatives of anthraquinones, there are only a few theoretical calculation studies for the  structural and spectral properties  [7-9]. There are reports that the  dihydroxy anthraquinones exhibit a  strong intramolecular hydrogen bond [7, 10]. Danthron is one of the simplest anthraquinone derivatives that shows the chromophore framework which is specific to many compounds of pharmaceutical and biological interest.

Theoretical studies such as the geometrical op-timization and vibrational spectra calculation of the drugs provide a very good insight for the changes in the conformation [11], reactive nature [12], relationship between the structures and the function [13], and the way in which the drugs interact with other molecules [14]. Previously the ground state geometry optimization used to be done by ab initio Hartree-Fock (HF) method. But since many years the density functional theory (DFT) method is the method of choice for the optimization of geometries. In the present study, the dihydroxy anth-raquinone derivatives danthron and emodin were optimized by ab initio HF and DFT using different basis sets.

To the  best of our knowledge, the  theoretical IR spectra calculation by DFT using a TZVP basis set, its correlation to experimental IR spectra and the assignments of the calculated vibrational modes of aromatic anthraquinone rings of danthron and emodin have not been reported so far.

The objective of this study is to do the assignments of the  vibrational modes obtained from the calculated IR spectra for both the molecules and correlate them with the experimental FTIR spectra. The experimental Raman spectra were also carried out for a  comparison purpose. The  study further focuses on the  calculations of HOMO-LUMO molecular orbitals of danthron and emodin. For knowing the assignments of IR spectra the vibrational frequency calculations are very helpful [15]. Therefore the study gives an insight to the different vibrational assignments of the calculated IR spectra of danthron and emodin.

2. Materials and methods

2.1. Computational details

The xyz coordinates of the dihydroxy anthraqui-none derivatives were obtained using the  ArgusLab software package [16]. The TmoleX quantum chemical calculation program was used to optimize the ground state geometries using the xyz coordinates [17]. Ground state geometric optimi-zations were performed at HF and DFT (B3LYP) levels. The def-SV(P) basis set was used for HF calculations, whereas the def-SV(P) as well as the triple zeta basis set (TZVP) were used for the DFT calculations for the  optimization of ground state geometries. The optimized geometries of danthron and emodin were used to calculate the vibrational spectra by the  DFT method. The  DFT (B3LYP) meth od with the  TZVP basis set was used for the vibrational (IR) spectra calculation, because IR frequencies are efficiently reproduced by the DFT method  [18]. The  calculated IR frequencies were corrected using a scaling factor of 0.9778. The assignments of the calculated vibrational frequencies of different vibrational modes along with ring vibrations were carried out based on visual observations using the software TmoleX which is a graphical user interface to the  TURBOMOLE quantum chemistry program package  [17]. The  visual observation provides an insight of the ring vibrations along with different vibrations of bonds. The soft-ware TmoleX was also used for visualizing the opti-mized geometry and the HOMO and LUMO orbitals of the optimized molecules.

2.2. Experimental methods

Danthron and emodin were procured from Sigma-Aldrich and used as such without any further purifi-cation. The infrared spectra of these molecules were recorded using an FTIR spectrometer (JASCO FT/ IR-6300). A resolution of 4 cm^-1 was used by the FTIR spectrometer while recording the spectra. The powder samples of the molecules were mixed separately with KBr to prepare the pellets using the pellet maker (Technosearch Instruments). The pellets were kept in the sample holder of the spectrophotometer and the FTIR spectra were recorded.

The Raman spectra were recorded using a home-built micro-Raman spectrometer. For recording the Raman spectra of danthron and emodin, a laser beam of 785 nm wavelength (Starbright Diode Laser, Torsana Laser Tech, Denmark) was focused onto the  samples by the  microscope objective. The laser beam with this particular wavelength was utilized to avoid the laser induced photo-damage of the sample by shorter wavelengths than this one. Also a longer wavelength helps to reduce the fluorescence effect which can compete with weak Ra-man signals. But longer wavelengths than this one will reduce the  detection efficiency  [19, 20]. The spectra were recorded using a 60X microscope objective (Nikon, Plan Fluor) with a 0.85 numerical aperture (NA). The spectral resolution of the spectrometer was 5.7 cm^-1 (with the spectrometer slit width kept at 100 µm) by measuring the FWHM of a 997 cm^-1 Raman band of the polystyrene bead spectrum.

The UV-Vis spectrum was recorded using a UV-Visible spectrophotometer (JASCO). 1 mg of danthron and emodin samples was mixed with 10 ml methanol separately to produce the stock solution. Then 0.1 ml of these solutions was diluted separately with 2.9 ml of methanol before recording the UV-Vis spectra for both the samples.

3. Results and discussion

3.1. Theoretical calculations

3.1.1. Geometry of danthron and emodin

Different ground state optimized conformations obtained for danthron and emodin using HF and DFT (BP86 and B3LYP) methods show that the  conformations are similar in different methods and basis sets. It is also noticed that all the  converged optimized conformations of dan-thron and emodin are completely planar in nature. The geometric optimization of danthron (1,8-di-hydroxy anthraquinone) and emodin (1,3,8-tri-hydroxy-6-methyl anthraquinone) by the  DFT (B3LYP) method with the  def-TZVP basis set produced three and eight conformations, respectively (Appendix, Tables A1 and A2). Conformation 1 (Conf-1) of danthron and emodin with two intramolecular hydrogen bonds have the  lowest total energy and so the most stable conformers as shown in Fig. 1.

The lowest total energy was always shown by the  conformation having the  double intramolecu-lar hydrogen bonding by the hydroxyl groups of C1 and C8 with the oxo group of C9. This result correlates with the results of Markovic et al. and Ruifa Jin et al. [7, 10]. The total energies obtained by HF and DFT with different basis sets for danthron explains that the  lowest total energy is produced by the conformation 1 with double intramolecular hydrogen bonds. The relative energies by HF and DFT (BP86) with different basis sets clarify that the conformation 1 with one intramolecular hydrogen bond is more stable compared to the  conformation 3, which is without any intramolecular hydrogen bond. When the total energies obtained from DFT (BP86) with def-SV(P) and TZVP basis sets were compared, it was clearly evident that the TZVP basis set produces lower total energy as compared to the  def-SV(P) basis set for a particular conformation.

When emodin was geometrically optimized at HF and DFT (BP86) levels, eight different conformations were converged. These structures are shown in Appendix, Table A2. The conformations 1, 2, 3 and 4 are closely similar to the conformations 5, 6, 7 and 8, respectively, except the orientation of hydroxyl group on C3.

The total energies show that the lowest total energy is obtained by the conformation 1 using DFT (BP86) with def-SV(P) and TZVP basis sets. The relative energies obtained by using DFT (BP86) with def-SV(P) and TZVP basis sets for the conformation 5 are 0.26 and 0.32 kcal/mol, respectively. But the HF with the def-SV(P) basis set indicates that the conformation 5 has the lowest energy, and the relative energy for the  conformation 1 is 0.08  kcal/mol, which is very less when compared with the relative energies produced between the  conformations 1 and 5 by DFT. Both the conformations 1 and 5 have double intramolecular hydrogen bonds. The relative energies specify that the conformations 2, 4, 6 and 8 have a  similar stability with one intramolecular hydrogen bond and the conformations 3 and 7 are the most unstable conformations without any intra-molecular hydrogen bonding.

3.1.2. HOMO and LUMO orbitals in danthron and emodin

In all the conformations of danthron the Huckel's method generated 268 molecular orbitals, out of which 62 were occupied and the rest were unoccupied molecular orbitals. The HOMO and LUMO were calculated by TmoleX using a 3D visualizer. The HOMO and LUMO for the conformation 1 of danthron are shown in Fig. 2(a, b).

When the  ground state geometry of emodin was optimized, a  total of 300 molecular orbitals were calculated by Huckel's method, out of which only 70 were occupied. The HOMO-LUMO were calculated for emodin by using the 3D visualizer of TmoleX. The HOMO and LUMO for the conformation 1 of emodin are shown in Fig. 3(a, b).

3.2. Experimental investigation of danthron

3.2.1. UV-Vis spectrum

Theoretical UV-Vis spectrum calculation was done using the  DFT method with B3LYP and the def-SVP basis set. For the theoretical calculation the conformation with the lowest total energy was used. The  calculated UV-Vis spectrum and the  experimental UV-Vis spectrum of danthron are shown in Fig. 4(a, b).

The theoretical and experimental UV-Vis spectra of danthron correlate well with each other.

3.2.2. FTIR spectrum of danthron

The FTIR spectrum for danthron is shown in Fig. 5 along with the  calculated IR spectrum of the most stable optimized geometry for danthron. From the correlation between the theoretical and the  experimental IR spectra, we observed that the calculated IR spectra of the conformer 1 with double intra-molecular hydrogen bonding correlated well with the  experimental IR spectrum whereas the peaks of IR spectrum from the conformer-3 deviate mostly from the peaks of the experimental FTIR spectrum (Appendix, Figs.  A1 and A2). Earlier the  IR spectrum has been reported by Smulevich  et  al.  [21]. In their work Smulevich et al. have explained two forms of dan-thron and for the IR frequency assignments they have given the approximate descriptions. In this work after optimizing the geometries for three different conformations their respective IR calculations were done. In our FTIR spectrum a strong and a  medium peak were observed at 783 and 456 cm^-1, respectively, which were not present in the previous study. The theoretical calculation correlates these bands with a peak at 782 and 460 cm^-1. The frequency assignment from the theoretical calculation suggested out-of-plane ring deformations for both the bands (Table 1).

3.2.3. Raman spectrum of danthron

Previously the Raman spectrum of danthron was reported by Fabriciova et al. [22]. For the comparison purposes we have also recorded the Raman spectrum of danthron which is shown in Fig. 6. As compared to the  previous work by Fabricio-va et al., we have successfully obtained a greater number of bands than before, i.e. 25 bands vs 16 bands before  [22] in the  range 1800-400  cm^-1. Among the  few new peaks which are present in this Raman spectrum of danthron, the  peaks at 1163 and 1341 cm^-1 have a strong intensity. Similarly the new bands observed at 741 and 781 cm^-1 show a medium intensity. One new weak peak at 1619 cm^-1 is observed in this present study which corresponds to the key characteristic very strong FTIR band of danthron at 1626 cm^-1.

3.2.4. Vibrational assignments

The theoretical IR calculations of danthron by the  DFT (B3LYP) method with the  basis set TZVP produced 72 different vibrational modes. Among the  72 vibrational modes, 24 were out-of-plane modes and the rest were in-plane ones. But in the previous work by Smulevich et al. only three vibrations were assigned as out-of-plane and many assignments were not defined. Here in this work we have mentioned the assignments about all the 72 theoretically obtained IR vibrational frequencies which are shown in Table 1. In Table 1, we have focused to present the theoretical IR vibrational modes with the  assignments and their correlation with the corresponding experimental wavenumbers. The  previous experimental works on Raman spectra and IR spectra are also mentioned along with the bands obtained in this present work in Table 1.

While visualizing the  vibrational modes by the  TmoleX program for assignments, we observed different vibrations of the  rings of dan-thron molecule such as stretching, breathing and deformation of rings. In the  previous studies, many assignments were mentioned as asymmetric deformity and skeletal deformity. But in our study we have given the complete assignment list with the specification of ring and different individual bond vibrations. Mixtures of both the  in-plane and out-of-plane vibrational modes were noticed in a  range of 973-45  cm^-1. All the  other vibrational modes were in-plane in a  range of 3296- 1032 cm^-1. Ring deformation was observed in vibrational modes of 208, 314, 335, 443, 447, 1080, 1213, 1270, 1361, 1450 and 1477 cm^-1. In 19 vibrational modes ring breathing was observed, out of which six modes were symmetric ring breathing and thirteen were asymmetric ring breathing. The  stretching of both the  O-H bonds was noticed for only two vibrational modes of 3267 and 3296  cm^-1. The  vibrational modes in a  range of 3109-3142 cm^-1 show mild stretching of few C-H bonds. The vibrational modes that were in a range of 3296-1700 cm^-1 produced mainly vibration involving either one single ring or specific bonds without involving any of the rings. The details of all the theoretical vibrational modes of danthron are mentioned in Appendix, Table A3.

γ: out of plane vibrational mode, Sym: symmetric and Asy: asymmetric.

3.3. Experimental investigation of emodin

3.3.1. UV-Vis spectrum

Theoretical UV-Vis spectrum calculation was done using the DFT method with B3LYP and the def-SVP basis set. For the theoretical calculation the conformation with the lowest total energy was used. The calculated UV-Vis spectrum and the  experimental UV-Vis spectrum of emodin are shown in Fig. 7.

The theoretical and experimental UV-Vis spectra of danthron correlate well with each other.

3.3.2. FTIR spectrum of emodin

From the  correlation between the  theoretical and the experimental IR spectra, we observed that the calculated IR spectrum of the conformer 1 with double intra-molecular hydrogen bonding correlated well with the experimental IR spectrum. The comparison graph is provided in Appendix, Figs. A3 and A4. The FTIR spectrum for emodin along with the calculated IR spectra of conformer 1 is shown in Fig. 8. The IR spectra of conformations 4 and 6 having one intra molecular hydrogen bond also correlate with the experimental IR spectrum. But when we compared the IR spectra along with the total energy of the  conformations, the  conformation 1 with two intra molecular hydrogen bonds has the most stable geometry and correlations to the experimental IR spectrum. Previously the IR and Raman spectra have been reported by Edwards et al. [23]. In their purely experimental work they have shown the IR and Raman spectra of emodin and mentioned the  peaks for both the  spectra with approximate vibrational assignments. In this present work after optimizing the  geometries for eight different conformations of emodin, their respective IR spectra calculations were done. We were able to observe a total of 38 peaks in the IR spectrum of emodin as compared to 22 peaks observed by the previous work in the 1800-400 cm^-1 range. Two peaks out of the 16 new peaks at 1033 and 1594 cm^-1 are found to have a strong intensity with out-of-plane ring deformation and a symmetric ring breathing type of vibrations, respectively. Similarly four new peaks at 418, 452, 546 and 648 cm^-1 have medium intensity levels with ring deformation (γ mode), asymmetric ring breathing, asymmetric ring stretching and ring deformation (γ mode) types of vibrations, respectively.

3.3.3. Raman spectrum of emodin

Previously the Raman spectrum of emodin was reported by Edwards et al. [23]. For the comparison purposes we have also recorded the  Raman spectrum of emodin which is shown in Fig. 9. We have successfully obtained a greater number of bands, i.e. 39 bands as compared to 26 bands [23] in the range 1800-400 cm^-1. Out of the 13 new bands one band, i.e. at 456 cm^-1, has a very strong intensity and correlates with the FTIR band of danthron at 452 cm^-1 with an asymmetric ring breathing type of vibration. Two other bands at 549 and 1590 cm^-1 show medium intensity levels that correlate with the FTIR bands at 549 and 1587 cm^-1 having the corresponding assignments of ring deformation (γ mode) and symmetric ring breathing, respectively.

3.3.4. Vibrational assignments

The theoretical IR calculation of the emodin molecule by DFT (B3LYP) method with the basis set TZVP produced a  total of 84 vibrational modes. The  frequency assignments were given for all the 84 vibrational modes, whereas in the previous work done by Edwards et al. [21] only 21 approximate frequency assignments were mentioned for the obtained IR spectrum. Out of the 84 vibrational modes obtained in the present work, the numbers of in-plane and out-of-plane vibrational modes are 43 and 41, respectively. This means that almost 50% of the vibrational modes are out-of-plane that were not found in the case of danthron. In the case of danthron one third of the total vibrational modes were out-of-plane. The increase in the number of out-of-plane vibrational modes may be attributed to the extra two functional groups (OH and CH₃) present in emodin in comparison to danthron.

The  theoretical IR values with frequency assignments and the  experimental IR values are mentioned in Table 2. Correlation has been done between both the values obtained in the present work along with the previously done work in Table  2. The  vibrational modes for each frequency were visualized using the TmoleX for their assignments and that revealed that the ring structure of emodin molecule was exhibiting stretching, deformation, symmetric and asymmetric breathing types of vibrational modes. In a frequency range of 904-30  cm^-1, both in-plane and out-of-plane types of vibrations were observed. But after that in a range of 3717-904 cm^-1 mostly in-plane vibrational modes were observed except three vibrational modes at 1033, 1452 and 3014  cm^-1. Ring deformation vibrational modes were both in-plane and out-of-plane. As compared to dan-thron, the number of asymmetric ring breathing vibrational modes in emodin is greater, i.e. 13 vs 27. The symmetric ring breathings are always in-plane vibrational modes both in danthron and emodin. The  two characteristic vibrational frequencies for danthron at 1624 and 1689 cm^-1 correlate with the bands in emodin at 1628 and 1689  cm^-1 having symmetric and asymmetric ring breathing, respectively. But the  FTIR spectra of danthron and emodin show that there is a  shift of the  band of 1675  cm^-1 in danthron to the 1666 cm^-1 band of emodin. This shift can be due to the  presence of the  extra two functional groups in emodin as compared to danthron. The stretching of mainly OH groups is observed at 3241, 3273 and 3717 cm^-1. Like the vibrations of danthron, the vibrational assignments for emo-din in the  range 3717-1689  cm^-1 also produce the vibrations either in one single ring or in some specific bonds only. The details of all the theoreti cal vibrational modes of emodin are given in Appendix, Table A4.

∗γ: out-of-plane vibrational mode, Sym: symmetric and Asy: asymmetric.

4. Conclusions

Our theoretical IR calculation studies were performed using the  most stable conformations of danthron as well as emodin having the  double intramolecular hydrogen bonding. The IR spectra calculated using the DFT (B3LYP) with the def-TZVP basis set relate well with the respective experimental FTIR spectra. Theoretically frequency assignments were allocated for all the vibrational modes. It was observed that there was a clear correlation between the  FTIR and Raman spectra with the  corresponding theoretical IR spectrum of the  most stable conformer. The  experimental FTIR and Raman spectra of danthron and emo-din show more bands as compared to the previously reported literature data. Some of the newly obtained bands with strong intensity levels are of great importance for the future database. The frequency assignments of danthron and emodin clearly mention the vibrational modes whether in-plane or out-of-plane. Also there is clarity regarding the types of ring vibrations of the molecules. These detailed studies of frequency assignments on the molecules will be very helpful for the future studies.

Acknowledgements

The authors are thankful to the FIST program of the  Government of India (SR/FST/PSI-174/2012) and the Department of Biotechnology (DBT), Government of India (BT/PR6413/MED/14/802/2005), for the facilities used in the work.

Appendix

Table A1. Different conformations of danthron with their total energy (TE) and relative energy calculated at HF and DFT (BP86) level with def-SV(P) and TZVP basis set.

Method
HF with def-SV(P) basis set	TE (Hartree)	-833.620029	-833.638059	-833.600770
	Relative energy (kcal/mol)	11.31	0	23.39
DFT (BP86)with def-SV(P) basis set	TE (Hartree)	-838.656440	-838.678801	-838.629784
	Relative energy (kcal/mol)	14.03	0	30.76
DFT (BP86) with TZVP basis set	TE (Hartree)	-839.566836	-839.587766	-839.541888
	Relative energy (kcal/mol)	13.13	0	28.79

Table A2. Different conformations of emodin with total energy (TE) and relative energy calculated at HF and DFT (BP86) level with def-SV(P) and TZVP basis sets.

Method
HF with def-SV(P) basis set	TE (Hartree)	-947.439852	-947.420929	-947.400895	-947.421061	-947.439977	-947.421061	-947.398713	-947.419650
	Relative energy (kcal/ mol)	0.08	11.95	24.52	11.87	0	11.87	25.89	12.75
DFT (BP86) with def-SV(P) basis set	TE (Hartree)	-953.137295	-953.114130	-953.087037	-953.113843	-953.136877	-953.113843	-953.084647	-953.112100
	Relative energy (kcal/ mol)	0	14.54	31.54	14.72	0.26	14.72	33.04	15.81
DFT (BP86) with TZVP basis set	TE (Hartree)	-954.184127	-954.162526	-954.136982	-954.162130	-954.183612	-954.162130	-954.134635	-954.160383
	Relative energy (kcal/ mol)	0	13.55	29.58	13.80	0.32	13.80	31.05	14.90

β: in-plane bending.

References

DANTRONO IR EMODINO MOLEKULINĖS SANDAROS IR SPEKTRŲ TYRIMAI FTIR, RAMANO SPEKTROSKOPIJOS IR TANKIO FUNKCIONALO METODAIS