Anti-Inflammatory and Analgesic Effects of the Marine-Derived Compound Excavatolide B Isolated from the Culture-Type Formosan Gorgonian Briareum excavatum
Authors: Sung-Chun Lin
Yin-Di Su
Ping-Jyun Sung
Yen-You Lin
Pei-Chin Chen
Chang-Yih Duh
Yen-Hsuan Jean
Zhi-Hong Wen
San-Nan Yang
Chien-Wei Feng
Chi-Min Li
Journal: Marine Drugs (2015)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: marine-derived compounds; diterpenes; anti-inflammatory activities; excavatolide B; proinflammatory mediators
Affiliations: Department of Marine Biotechnology and Resources, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 80424, Taiwan. ., Department of Orthopaedic Surgery, Ping-Tung Christian Hospital, Ping-Tung 90059, Taiwan. ., Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 80424, Taiwan. ., National Museum of Marine Biology & Aquarium, Pingtung 94450, Taiwan. ., School of Medicine, College of Medicine and Department of Pediatrics, E-DA Hospital, I-Shou University, Kaohsiung 84001, Taiwan. ., Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 94450, Taiwan. .

Abstract:  In recent years, several marine-derived compounds have been clinically evaluated. Diterpenes are secondary metabolites from soft coral that exhibit anti-inflammatory, anti-tumor and cytotoxic activities. In the present study, we isolated a natural diterpene product, excavatolide B, from cultured Formosan gorgonian Briareum excavatum and investigated its anti-inflammatory activities. We found that excavatolide B significantly inhibited the mRNA expression of the proinflammatory mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-challenged murine macrophages (RAW 264.7). We also examined the anti-inflammatory and anti-nociceptive effects of excavatolide B on intraplantar carrageenan-induced inflammatory responses. Excavatolide B was found to significantly attenuate carrageenan-induced nociceptive behaviors, mechanical allodynia, thermal hyperalgesia, weight bearing deficits and paw edema. In addition, excavatolide B inhibited iNOS, as well as the infiltration of immune cells in carrageenan-induced inflammatory paw tissue. 

Retracted: No

PubPeer comments:
 The content of this research mainly came from part of the Master degree thesis of Chi-Min Li, the 6th author of this article, who graduated in 2011 from Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan. From the abstract of this thesis, it is quite obvious that the compound used in this investigation, excavatolide B, was isolated from the marine organism Briareum excavatum inhabiting in ocean, and not from the culture-type one as shown in the article. Please see the abstract of the Master degree thesis of Chi-Min Li as shown below. ABSTRACT: In recent years, studies have increasingly recognized that many natural products with biological activity have been isolated from marine organisms, while the chemical structures are very different from those of land-based organisms. Therefore, the ocean is a natural drug source. Regarding drug screening, anti-inflammatory activity has become a key point, and many studies confirm that inflammation plays an important role in many human diseases. Many different compounds are now in the clinical evaluation stage. However, the inflammation-related diseases being closely linked, there is an urgent need to study the anti-inflammatory effects as well as screen the therapeutic drugs for research and development. In this study, we isolated and purified compounds from Formosan gorgonian (Briareum excavatum) and Formosan soft coral (Lobophytum sarcophytoides) and investigated biological activities. We confirmed that the natural compound Brei from B. excavatum and the compounds Sac-1 and Sac-2 from L. sarcophytoides produced significant inhibition of the proinflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced murine macrophages (RAW 264.7) cell model. We examined in vivo whether the B. excavatum Brei has anti-inflammatory and antinociceptive effects by using the carrageenan-induced inflammation model. Using the paw-edema assay, we performed several important investigations such as the plantar analgesia test, mechanical hyperalgesia test (allodynia), and weight-bearing analysis of animal behavior to evaluate the degree of pain and inflammation. Our results demonstrate that the natural product Brei can reduce paw-pad swelling, thermal hyperalgesia, threshold latency, and improve the affected limb in the carrageenan-induced inflammatory model. In the histopathology analysis, we showed that Brei significantly inhibited the aggregation and infiltration of inflammation-related blood cells and improved the inflammatory status of the tissues. Therefore, the marine natural compound Brei has anti-inflammatory activity and it can be used as a therapeutic compound for acute inflammation in the near future. , 
 Not all of the results came from the above thesis. The target compound (Exc-B) used in the study were obtained from (1) wild-type; and (2) cultured-type octocoral, B. excavatum, and the cultured-type organism was listed as representative species in the title of our research work. In this study, we focus on the biological activity and possible mechanisms of this compound. Thanks , 
 I sincerely hope that Zhi-Hong Wen, who is the Master degree thesis advisor of Chi-Min Li and the important author of this 2015 paper published in Marine Drugs, can carefully compare the content of the abstract of the paper with the partial content of the abstract of the Master degree thesis. After that, I think you might not insist on your words that  not all of the results came from the above thesis . Partial content of the abstract of the degree thesis is as following: In this study, we isolated and purified compounds from Formosan gorgonian (Briareum excavatum) and Formosan soft coral (Lobophytum sarcophytoides) and investigated biological activities. We confirmed that the natural compound Brei from B. excavatum and the compounds Sac-1 and Sac-2 from L. sarcophytoides produced significant inhibition of the proinflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced murine macrophages (RAW 264.7) cell model. We examined in vivo whether the B. excavatum Brei has anti-inflammatory and antinociceptive effects by using the carrageenan-induced inflammation model. Using the paw-edema assay, we performed several important investigations such as the plantar analgesia test, mechanical hyperalgesia test (allodynia), and weight-bearing analysis of animal behavior to evaluate the degree of pain and inflammation. Our results demonstrate that the natural product Brei can reduce paw-pad swelling, thermal hyperalgesia, threshold latency, and improve the affected limb in the carrageenan-induced inflammatory model. In the histopathology analysis, we showed that Brei significantly inhibited the aggregation and infiltration of inflammation-related blood cells and improved the inflammatory status of the tissues. Also, the compound excavatolide B was also named as Brei in the indicated thesis and this Marine Drugs paper. What is the compound Exc-B in your response? Is there any connection with this issue? , 
 Chi-Min Li showed that results of the graduate thesis solely came from excavatolide B (Brei) of wild-type coral (inhibiting in ocean), different to the opinion of Zhi-Hong Wen who claimed that part of excavatolide B used in this research was obtained from culture -type octocoral. Since the thesis published much early than the Marine Drugs article, authors need to reasonly explain, with the situation that Figures of animal studies are exactly identical in both thesis and journal article. Also, it is incredibe that the numbers of animal for each testing could be altered even from n= 3 in degree thesis of Li, to n= 6 in the paper published later. , 
 In page 2570,the preparation of the anti-inflammatory coral natural product excavatolide B was below described. 4.1. Preparation of Excavatolide B In the present study, excavatolide B (1R, 2S, 4S, 4aR, 5S, 6S, 8Z, 9aS, 12aS, 13R, 13aS)-4,5, 13-triacetoxy-2-hydroxy-1,4a,8,11a-tetramethyl-11-oxo-2,3,4,4a,5,6,7,9a,11,11a,13,13adodecahydro-1H benzo[4,5]cyclodeca[1,2-b]oxireno[c]furan-6-yl butyrate (Figure 1A) was isolated from Formosan gorgonian Briareum excavatum ............................................... . The organic extract was partitioned into H2O and ethyl acetate layers, and the ethyl acetate layer was separated further over a normal phase silica gel by column chromatography eluted with n-hexane, ethyl acetate and methanol to yield 30 fractions. Fractions 13u201316 was separated using a normal phase silica gel elution with n-hexane-ethyl acetate to yield a subfraction and then further purified by a normal phase silica gel elution with n-hexane-ethyl acetate (80:1) to acquire the pure compound, excavatolide B (2.13 g). The structure of the compound was confirmed by nuclear magnetic resonance spectroscopy. The purity ( 98%) of excavatolide B was evaluated and verified by 1H-NMR and 13C-NMR spectra (Varian Mercury Plus 400 FT-NMR at 400 MHz). Furthermore, the structure was also compared to that of natural compounds derived from soft coral, as described previously [31]. Two queries about the above procedure to yield excavatolide B were raised. 1.In the cited reference 31, excavatolide B was obtained from silica gel colume with elution of hexane ethyl acetate(1:3). However, in this study excavatolide B was obtained from a silica gel column with elution of a much less polar eluent hexane ethyl acetate(80:1). Is it possible to elute excavatolide B from the silica gel column using this very low polar eluent ? 2.Please show how the purity ( 98%) of excavatolide B was evaluated and verified by 1H-NMR and 13C-NMR spectra as described above. , 
 The #4 comment about the alteration of the numbers of animal for testing, and two questions of #5 comment about the procedure for isolation and purification of the bioactive compound excavatolide B (Brei) and the method to verify the purity( 98%) of excavatolide B by 1H-NMR and 13C-NMR spectra are needed to be clarified by authors . It should be not possible that excavatolide B could be isolated by a normal phase silica gel column with elution of a very low polar solvent n-hexane-ethyl acetate (80:1), as the cited reference 31 reported the isolation of 5 new compounds of similar structure type including excavatolide B, which were all isolated by elution with much polar eluent in comparison with the polarity of the eluent used in this study. Also, based to the known scientific knowledge it is nearly not possible to accurately measure the purity of organic compounds by 1H-NMR and 13C-NMR spectra, otherwise please advise. , 
 It seems that some studies have used the integration for signals in proton NMR to estimate the ratio for different compounds in a mixture, however, using 1H-NMR in precise quatification might be difficult as there are deviations in calculatihg the area of NMR peaks. But due to the rapid advancement of NMR technique, this technical problem might have been solved well. It is highly appreciated if authors can provide the latest information of this important issue. , 
 Part of the #2 comment from Zhi-Hong Wen, one of the authors,is as below :  Not all of the results came from the above thesis. The target compound (Exc-B) used in the study were obtained from (1) wild-type; and (2) cultured-type octocoral, B. excavatum, and the cultured-type organism was listed as representative species in the title of our research work. But by comparison of the 2011 thesis of C.M. Li and this Mar Drugs article, it seems that the initial study of inhibitory effect of excavatolide B towards iNOS and COX-2 protein expression in LPS-activated macrophage cells and most of the animal tests in this article have been accomplished in the 2011 thesis of Li. That means that the essential part of this work came from the target compound (Exc-B) obtained from wild-type marine organism from ocean, but not from the cultured-type organism . , 
 The principal author Zhi-Hong Wen should clarify the reason that why compound Exc-B used in this study actually came from wild-type soft coral, but was described as from the cultured-type one in this article, as one can see from the above comments, including #2 comment which is a response from Zhi-Hong Wen . Also, the numbers of animal for each testing was altered even from n= 3 in degree thesis of Li, to n= 6 in the this paper , as mentioned in #4 comment. , 
 This article reported that the purity ( 98%) of excavatolide B was evaluated and verified by 1H-NMR and 13C-NMR spectra. The authors should show the 1H-NMR and 13C-NMR spectra and the method to calculate the purity to confirm the purity of excavatolide B, as if excavatolide B is not in such a high purity, the claimed biological activities might be arisen from other minor compounds mixed with excavatolide B. , 
 ![file] Comments 1, 3 and 4 revealed that the content of this article mainly came from the Master degree thesis of Chi-Min Li, the 6th author of this article, who graduated in 2011 from Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan, and his thesis advisor Dr. Zhi-Hong Wen did not deny this in his reply (comment #2). The Fig.4 of this article ( published in 2015),seen in right-half column, was found to display the identical results as that found in Fig. 22 of the 2011 thesis of Chi-Min Li, however, the numbers of tested animal n=3 (control), n=5 (carrageenan + Exc-B 60 mg Kg), n=4 (carrageenan + Exc-B 15 mg Kg) and n=4 (carrageenan), were all altered to n=6 in this article. , 
 ![image] , 
 Careful comparison of the Fig. 21 and 22 of 2011 Master degree thesis of Chi-Ming Li in the left column of #11 and #12 with Fig. 4 (A) and 4(C) of this article, it was found that the experimental results of the group Exc-B(60 mg Kg, n=6) and the group carrageenan + indomethacin (5mg Kg, n=6) have not been mentioned in the 2011 thesis. How can the 2015 research article of this study show the presence of these results? , 
 ![file] , 
 Fig.4 B of this article ( published in 2015), seen in right-half column, was found to display the identical results as that found in Fig. 23 of the 2011 thesis of Chi-Min Li on the left column, however, the numbers of tested animal n=3 (control), n=5 (carrageenan + Exc-B 60 mg Kg), n=4 (carrageenan + Exc-B 15 mg Kg) and n=4 (carrageenan), were all altered to n=6 in this article. , 
 By careful examination between Fig. 23 of 2011 Master degree thesis of Chi-Min Li in the left column of #14 comment and Fig. 4 (B) of this article in the right column , it was found that not only the numbers of tested animal n=3 (control), n=5 (carrageenan + Exc-B 60 mg Kg), n=4 (carrageenan + Exc-B 15 mg Kg) and n=4 (carrageenan), were all altered to n=6 in this article which incrediblly generated exactly the same experimental curves as those in 2011 thesis, but also the experimental data of the two groups Exc-B(60 mg Kg, n=6) and carrageenan + indomethacin (5mg Kg, n=6) in this 2015 article were not mentioned in the 2011 thesis. How can the 2015 article of the same study give the data which were found to be absent in the original study? , 
 It was mentioned in subsection 4.1 Preparation of Excavatolide B in page 2570, that the purity ( 98%) of excavatolide B was evaluated and verified by 1H-NMR and 13C-NMR spectra (Varian Mercury Plus 400 FT-NMR at 400 MHz). I have the same question as those of #5,6 and 7 comments about the ability for using 1H-NMR in precise quantification might be very difficult as there are deviations in integrating the area of NMR peaks. Furthermore, till now the 13C-NMR spectroscopic technology can not be used for the ratio estimation of different carbons. Thus, 13C-NMR spectra are totally useless for evaluation and verification of the purity of the compound, , 
 Comments 5, 6 ,7 and 17 are all showing suspicious on the use of 1H-NMR and 13C-NMR to precisely analyze and measure the purity of a mixture. My previous experience is that the integration of 1H-NMR peaks could be used to estimate the approximate ratio of different compounds in the mixture by comparison of peak area from integration of the corresponding proton peaks of different compounds in the mixture. However, it is very difficult to accurately calculate the percent purity of a compound in a mixture. If nowadays the purity of compound in a mixture could be precisely quantified by more advanced 1H- NMR and 13C-NMR technology, please tell the literature of this new knowledge so that more peers can be beneficial from using this new technology to solve scientific problem. , 
 #17 13C NMR can be used for quantitation or for the estimation of the ratio of two or more compounds, but the standard 13C pulse sequence (or its parameters) needs to be modified, and the sample spectrometer have to meet certain requirements, specifically: - {1H} decoupling needs to be disabled during the relaxation phase of the pulse sequence to avoid the buildup of the nuclear Overhauser effect (which would ultimately affect the intensities of 13C signals depending on the proximity of the carbon nuclei to hydrogen nuclei in molecules) - if this is not done, then a massive error can occur when integrating, let us say, a carbon from a methyl group and a quaternary carbon; - the relaxation delay needs to be set to several tens of seconds as 1.) the T1-relaxation time of 13C nuclei is fairly long, especially for quaternary carbons, and 2.) the {1H} decoupling during acquisition also generates the nuclear Overhauser effect - after waiting a few tens of seconds the effect should dissipate; - the excitation pulse should have a good enough excitation profile so that all 13C signals throughout the fairly large scale of resonance frequencies (in Hz) are affected by the pulse the same way; - the signal-to-noise ratio should be as high as possible In my experience, measuring a good and reliable quantitative 13C spectrum with low signal-to-noise ratio is not easy. If any quantitation without the first two steps listed above is attempted, I would guess that the quantitation would be very inaccurate. The estimation of the purity of the sample by 1H is also tricky, especially if one desires to obtain the purity as a mass percent - if so, one would need to identify all impurities in order to know their molar mass and to correctly calculate such purity. What I personally miss in the paper are the spectra of the studied compound, or at least its 1H and 13C NMR data report - especially if the authors state that the compound s structure was confirmed by nuclear magnetic resonance. Of course, the journal might have not required such data. , 
 Please see content of the page 2572 of this article. 4.7. Animal Experimental Design Male Wistar rats (n = 30) were randomly divided into six groups: control (0.5 mL saline with 20% DMSO, n = 6), 1.5% carrageenan + vehicle (n = 6), 1.5% carrageenan + 15 mg kg excavatolide B (n = 6), 1.5% carrageenan + 60 mg kg excavatolide B (n = 6), 1.5% carrageenan + 5 mg kg indomethacin (n = 6) and 60 mg kg excavatolide B. The number of male Wistar should be 36, not 30, as the 6th group 60 mg kg excavatolide B should also be carried out with n =6, too. ,