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Cite This: J. Agric. Food Chem. 2018, 66, 4581−4583
Critical Problems Stalling Progress in Natural Bioactive Polysaccharide Research and Development Quan-Bin Han* School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon Tong, Hong Kong, People’s Republic of China ABSTRACT: Natural polysaccharides are attracting increasing attention from food and pharmaceutical industries for their wide range of valuable biological activities. However, the poor repeatability of the methods used in sample preparation and chemical characterization is hampering both research and product development. The unstandardized quality, in turn, undermines efforts to understand the mechanism by which they work via oral dose, which is essential to realize the full beneficial potential of polysaccharides. Some scientists believe polysaccharides work by direct gut absorption; however, increasing evidence points to the gut microbiome and intestinal Peyer’s patches as holding the keys to how they work. KEYWORDS: polysaccharide, quality control, method repeatability, bioavailability, phagocytosis
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structure characterization. Take Ganoderma lucidum as an example, which is one of the mostly studied mushrooms. Although there are hundreds of research articles reporting the polysaccharides,4,5 none of them stated that their polysaccharides are identical to any previously reported. It is because the repeatability of the separation method and the identification method is so poor that people are not able to confirm whether or not the polysaccharides that they are studying are exactly the same as any reported. The separation methods that we are using decide that we are not able to isolate two completely identical polysaccharides from natural resources. Unlike small molecules, polysaccharides naturally exist as a mixture; a single polysaccharide molecule has never been obtained from natural resources. Separation and purification of a polysaccharide from food materials always involve a complicated procedure, in which water extraction, ethanol precipitation, and deproteination might be the commonly used operation before repeated chromatographic purification. Take ethanol precipitation as an example. The response of polysaccharide precipitates to the ethanol concentration varies greatly in terms of the precipitate yield and molecule size as a result of the diverse chemical structures of their components.6 Not to say that the ethanol concentration used here has never been fixed to an accurate value as 80.15 or 70.06%. Now chromatography has been popularly used in separation of polysaccharides because many new separation materials are adopted and the polysaccharide fractions could be greatly purified, similar to what we did to small molecules. However, the outcome of chromatographic fractionation is affected by many factors, while repeatability is one of the basic validation criteria for chromatography. Even the same crude polysaccharide sample, separated on the same separation material, in the
olysaccharides isolated from natural sources are attracting increasing attention from food and pharmaceutical industries, because some of them exhibit a variety of biological activities, such as anticancer, antiviral, anti-inflammatory, and immunostimulatory activities.1−3 In comparison to smallmolecule-based drugs and food supplements, these bioactive polysaccharides from edible materials are generally safer, more effective with fewer side effects, and more readily available if not cheaper. However, two major concerns stall the research and development progress in natural bioactive polysaccharides: quality standardization and mechanism of action. For research to make progress and for industry to develop products, standardization is necessary. The identity of the substances being used must be quantified and characterized, so that researchers can build on results from each other and so that industry can develop systems of quality control. Unfortunately, few publications stated that the purified and characterized polysaccharides had been isolated previously by other research teams. In other words, the purification and characterization of polysaccharides from natural sources are hard to repeat, and poor repeatability makes quality control very difficult. Another big concern is the mechanism of action of these bioactive polysaccharides. As a kind of highly polar macromolecule, if not digested into mono/oligosaccharides, many bioactive polysaccharides are absorbed very poorly when ingested orally; however, this is the primary way they are usually taken, and this is the way that they exert their various and significant effects. Little is known about precisely how these orally dosed polysaccharides act, yet understanding the mechanism is critical to both research and pharmaceutical product development.
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CONCERN ON THE QUALITY STANDARDIZATION OF POLYSACCHARIDES Currently, developing standards for the quality control of polysaccharides is hindered by one major difficulty: poor repeatability of the methods used for sample preparation and © 2018 American Chemical Society
Received: Revised: Accepted: Published: 4581
January 26, 2018 April 1, 2018 April 16, 2018 April 16, 2018 DOI: 10.1021/acs.jafc.8b00493 J. Agric. Food Chem. 2018, 66, 4581−4583
Journal of Agricultural and Food Chemistry
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same column, eluted with the same mobile phase and sample collection procedure, has been known to produce inconsistent results if the same operator combined the fractions slight differently, because the isolates are still mixtures and the composition of the mixtures varies and depends upon the natural materials, extraction methods, column type/size/flow rate/detection, and operators. Because each fraction is a specific mixture, a new mixture will be generated once different fractions are combined together. When identical samples cannot be prepared reliably, research work cannot be repeated nor generalized and industry cannot create standardized products. Even if the sample preparation could be reliably repeated, the subsequent structure characterization involves more complicated operations and also has a bigger concern of poor repeatability in methodology. The results of molecular size analysis varied greatly between different analytical methods; even for the same high-performance gel permeation chromatography (HPGPC) method, different analytical columns and different reference standard polymers also caused a big difference. In some cases, the sugar composition was directly determined only by integrating the peak area without any standard references. Methylation analysis has been popularly used to indicate the sugar linkage composition,7 but the repeatability is so poor that no one has exactly repeated the results of any study. The normal methylation analysis protocol mainly has seven steps: methylation plus dialysis, commonly performed twice (some need more), hydrolysis, reduction, and acetylation, followed by gas chromatography−mass spectrometry determination,7 and all of these are variable. Assuming that every dialysis operation and chemical reaction generates an acceptable relative standard deviation (RSD) of 5%, the final RSD could be as large as 40%. All of these above-mentioned variations are enough to create different characterization results. Taken together, the poor repeatability of methods used in sample preparation and chemical characterization make the quality control of highly diverse polysaccharides a challenge and concern. This, in turn, greatly limits the research and development of polysaccharide-based products. Continuous efforts are being made to solve this quality control problem. In the Chinese Pharmacopoeia, the characteristics of Dendrobium polysaccharides are described in terms of a monosaccharide profile: only mannose and glucose in a ratio of 2.8:1−8:1.8 However, this method fails to distinguish individual Dendrobium species because all of them present an identical monosaccharide profile. To overcome this failure, a holistic polysaccharide marker Dendrobium officinale polysaccharide (DOP) has been successfully adopted in the qualitative and quantitative analysis of Dendrobii Officinalis Caulis.9 This polymer marker is unique and specific enough for qualitative authentication purposes, and the quantitation results are comparable to that of quantitative determination of the monosaccharide profile. However, this polysaccharide marker approach only fit a few cases because not all natural products contain such a special polymer marker9 and it may not work for formula products. Oligosaccharides, with structures somewhere between monomers and polymers, offer another option for a unique marker. Oligosaccharide markers might contain both the bioactive structural unit and a specific structural feature.10,11 Oligosaccharide mapping technology based on enzyme hydrolysis has been developed for authentication purposes.12−14
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CONCERN ON THE MECHANISM OF ACTION
In addition to the quality control issue, the lack of evidence regarding the mechanism of action is another major barrier to realize the full potential of polysaccharides in the modern world. How polysaccharides work is a true puzzle. Normally, polysaccharides are ingested orally, either in decoctions or as pills or capsules. As such, they are known to be poorly absorbed, yet they have important observable physiological effects. There are three theories as to their mechanism of action: direct absorption, via gut microbiota, or via Peyer’s patches. Although almost all of the current pharmacokinetic studies focused on the sample by intravenous administration, some scientists have made efforts to prove that polysaccharides are absorbed in the gastrointestinal tract. They have used various labeling reagents, especially fluorescent labeling, to increase detection sensitivity;15−20 however, none of these fluorescencelabeling methods themselves have been well-validated; therefore, results from using them may not be reliable. Did the polysaccharides, after being labeled, retain their original bioactivities and physical/chemical properties? Is it possible that the original polysaccharide was not absorbable but the labeling reagent changed the structure of the original polysaccharide such that it was then absorbed? Was the stability of the labeled polysaccharide determined? Did the labeled polysaccharide contain any free labeling reagent? Was the purity of the labeled polysaccharide checked using HPGPC? Without satisfactory validation of the analytical method, such absorption results are suspect. In addition to direct gut absorption, there are two other feasible mechanisms by which orally dosed polysaccharides can have significant impact. These are via gut microbiota or via Peyer’s patches. In the past 10 years, research on gut microbiota has flourished, and the interaction between polysaccharides and gut microbiota has been a focus of many studies.21 Xu et al. have reviewed the literature regarding the role of polysaccharides as prebiotics21 and offer convincing evidence that polysaccharides work by altering the species composition of the gut microbiome. Here, we propose another possibility, Peyer’s patches. It is hypothesized that polysaccharides, without the need to enter the blood/lymph circulation, trigger immune response immediately after entering Peyer’s patches, where they activate innate immune cells. A recent study has indicated that, after a 3 h journey in the small intestine, Angelica polysaccharide was quickly digested when it arrived in the large intestine.20 This means that the polysaccharide stays longer in the small intestine than anywhere else and suggests that the small intestine is where it exerts its effects. Peyer’s patches, as the main immune organ in the small intestine, are small masses of lymphatic tissue found throughout the ileum. These patches contain several groups of immune cells, such as T cells, dendritic cells, and macrophages, all of which are targets of polysaccharides.22−25 The question that next arises is how do these polysaccharides enter Peyer’s patches? In the study by Rice et al.,18 flow cytometry analysis of gut-associated lymphoid tissue (GALT) cells isolated from Peyer’s patches of mice gavaged with fluorescently labeled glucans revealed that GALT cells are capable of recognizing and binding glucans. Uptake and internalization of fluorescently labeled glucans by murine gastrointestinal epithelial cells were also observed using confocal imaging. However, the epithelial cells have not been 4582
DOI: 10.1021/acs.jafc.8b00493 J. Agric. Food Chem. 2018, 66, 4581−4583
Perspective
Journal of Agricultural and Food Chemistry
(9) Xu, J.; Li, S. L.; Yue, R. Q.; Ko, C. H.; Hu, J. M.; Liu, J.; Ho, H. M.; Yi, T.; Zhao, Z. Z.; Zhou, J.; Leung, P. C.; Chen, H. B.; Han, Q. B. A novel and rapid HPGPC-based strategy for quality control of saccharide-dominant herbal materials: Dendrobium of ficinale, a case study. Anal. Bioanal. Chem. 2014, 406, 6409−6417. (10) Zhao, L. Y.; Wu, M. Y.; Xiao, C.; Yang, L.; Zhou, L. T.; Gao, N.; Li, Z.; Chen, J.; Chen, J. C.; Liu, J. K.; Qin, H. B.; Zhao, J. H. Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 8284−8289. (11) Li, S. P.; Wu, D. T.; Lv, G. P.; Zhao, J. Carbohydrates analysis in herbalglycomics. TrAC, Trends Anal. Chem. 2013, 52, 155−169. (12) Wu, D. T.; Xie, J.; Hu, D. J.; Zhao, J.; Li, S. P. Characterization of polysaccharides from Ganoderma spp. using saccharide mapping. Carbohydr. Polym. 2013, 97, 398−405. (13) Guan, J.; Zhao, J.; Feng, K.; Hu, D. J.; Li, S. P. Comparison and characterization of polysaccharides from natural and cultured Cordyceps using saccharide mapping. Anal. Bioanal. Chem. 2011, 399, 3465−3474. (14) Wu, D. T.; Cheong, K. L.; Wang, L. Y.; Lv, G. P.; Ju, Y. J.; Feng, K.; Zhao, J.; Li, S. P. Characterization and discrimination of polysaccharides from different species of Cordyceps using saccharide mapping based on PACE and HPTLC. Carbohydr. Polym. 2014, 103, 100−109. (15) Yi, Y.; Wang, H.; He, J. Research progresses of pharmacokinetics of polysaccharides. Acta Pharmaceut. Sin. 2014, 49, 443−449. (16) Koyama, Y.; Miyagawa, T.; Kawaide, A.; Kataoka, K. Receptormediated absorption of high molecular weight dextrans from intestinal tract. J. Controlled Release 1996, 41, 171−176. (17) Vetvicka, V.; Dvorak, B.; Vetvickova, J.; Richter, J.; Krizan, J.; Sima, P.; Yvin, J. C. Orally administered marine (1−3)-beta-D-glucan Phycarine stimulates both humoral and cellular immunity. Int. J. Biol. Macromol. 2007, 40, 291−298. (18) Rice, P. J.; Adams, E. L.; Ozment-Skelton, T.; Gonzalez, A. J.; Goldman, M. P.; Lockhart, B. E.; Barker, L. A.; Breuel, K. F.; DePonti, W. K.; Kalbfleisch, J. H.; Ensley, H. E.; Brown, G. D.; Gordon, S.; Williams, D. L. Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge. J. Pharmacol. Exp. Ther. 2005, 314, 1079−1086. (19) Lin, X.; Wang, Z.; Sun, G.; Shen, L.; Xu, D.; Feng, Y. A sensitive and specific HPGPC-FD method for the study of pharmacokinetics and tissue distribution of Radix Ophiopogonis polysaccharide in rats. Biomed. Chromatogr. 2010, 24, 820−825. (20) Wang, K. P.; Cheng, F.; Pan, X. L.; Zhou, T.; Liu, X. Q.; Zheng, Z. M.; Luo, L.; Zhang, Y. Investigation of the transport and absorption of Angelica sinensis polysaccharide through gastrointestinal tract both in vitro and in vivo. Drug Delivery 2017, 24, 1360−1371. (21) Xu, J.; Chen, H. B.; Li, S. L. Understanding the molecular mechanisms of the interplay between herbal medicines and gut microbiota. Med. Res. Rev. 2017, 37, 1140−1185. (22) Zhang, J.; Tang, Q.; Zhou, C.; Jia, W.; Da Silva, L.; Nguyen, L. D.; Reutter, W.; Fan, H. GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response. Life Sci. 2010, 87, 628−637. (23) Kim, H. Y.; Kim, J. H.; Yang, S. B.; Hong, S. G.; Lee, S. A.; Hwang, S. J.; Shin, K. S.; Suh, H. J.; Park, M. H. A polysaccharide extracted from rice bran fermented with Lentinus edodes enhances natural killer cell activity and exhibits anticancer effects. J. Med. Food 2007, 10, 25−31. (24) Sun, L. X.; Lin, Z. B.; Li, X. J.; Li, M.; Lu, J.; Duan, X. S.; Ge, Z. H.; Song, Y. X.; Xing, E. H.; Li, W. D. Promoting effects of Ganoderma lucidum polysaccharides on B16F10 cells to activate lymphocytes. Basic Clin. Pharmacol. Toxicol. 2011, 108, 149−154. (25) Sun, L.-X.; Lin, Z.-B.; Duan, X.-S.; Lu, J.; Ge, Z.-H.; Li, X.-J.; Li, M.; Xing, E.-H.; Jia, J.; Lan, T.-F.; Li, W.-D. Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells. J. Pharm. Pharmacol. 2011, 63, 725−735.
specifically identified. Once identified, as with gut microbiota, there will be complete evidence that Peyer’s patches are intimately involved in the biochemical mechanism by which polysaccharides exert their effects.
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CONCLUSION AND SUGGESTIONS In summary, lack of quality control procedures for producing polysaccharides and lack of knowledge of the mechanism by which they work are delaying progress in the research and development of polysaccharide-based health products. Scientists need to reach consensus on purity and structural characterization and to determine reliable means to produce identical samples. As for the mechanism, while some scientists pursue the possibility of direct gut absorption, increasing evidence points to the gut microbiome and/or Peyer’s patches for solving the mystery of polysaccharide activity.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 00852-34112906. Fax: 00852-34112461. E-mail:
[email protected]. ORCID
Quan-Bin Han: 0000-0001-9051-1485 Funding
The author was financially supported by the Hong Kong Special Administrative Region (HKSAR) Innovation and Technology Fund (ITF), Tier 3, ITS/311/09, the General Research Fund (12100615 and 22100014), the Health Medical Research Fund (11122531 and 14150521), the National Natural Sciences Foundation in China (81473341), and the Hong Kong Baptist University [Research Committee (RC) Startup Grants MPCF-001-2014/2015, RC-IRMS/14-15/06, FRG1/16-17/032, and FRG2/16-17/002]. Notes
The author declares no competing financial interest.
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REFERENCES
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DOI: 10.1021/acs.jafc.8b00493 J. Agric. Food Chem. 2018, 66, 4581−4583
