Critical Problems Stalling Progress in Natural Bioactive

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Critical problems stalling progress in natural bioactive polysaccharide research & development Quan-Bin Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00493 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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Journal of Agricultural and Food Chemistry

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Critical problems stalling progress in natural bioactive polysaccharide research

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& development

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Quan-Bin Han

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School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China

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Abstract:

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Natural

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pharmaceutical industries for their wide range of valuable biological activities.

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However, the poor repeatability of the methods used in sample preparation and

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chemical characterization is hampering both research and product development. The

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unstandardized quality in turn undermines efforts to understand the mechanism by

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which they work via oral dose, which is essential to realizing the full beneficial

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potential of polysaccharides. Some scientists believe polysaccharides work by direct

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gut absorption; however, increasing evidence points to the gut microbiome and the

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intestinal Peyer’s patches as holding the keys to how they work.

polysaccharides are

attracting increasing attention from food

and

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Keywords: Polysaccharide, quality control, method repeatability, bioavailability,

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phagocytosis 1

ACS Paragon Plus Environment


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Polysaccharides isolated from natural sources are attracting increasing attention from

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food and pharmaceutical industries, because some of them exhibit a variety of

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biological

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immunostimulatory.1-3 Compared to small molecule-based drugs and food

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supplements, these bioactive polysaccharides from edible materials are generally safer,

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more effective with fewer side effects, and more readily available if not cheaper.

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However, two major concerns stall the research & development progress in natural

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bioactive polysaccharides: quality standardization and mechanism of action.

activities

such

as

anticancer,

antiviral,

anti-inflammatory,

and

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In order for research to make progress and for industry to develop products,

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standardization is necessary.

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quantified and characterized, so that researchers can build on each other’s results and

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so that industry can develop systems of quality control. Unfortunately, few

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publications stated that the purified and characterized polysaccharides had been

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isolated previously by other research teams. In other words, the purification and

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characterization of polysaccharides from natural sources are hard to repeat, and poor

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repeatability makes quality control very difficult.

The identity of the substances being used must be

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Another big concern is the mechanism of action of these bioactive polysaccharides.

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As a kind of highly polar macromolecule, if not digested into mono-/oligo-saccharides,

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many bioactive polysaccharides are absorbed very poorly when ingested orally;

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however, this is the primary way they are usually taken—and this is the way they

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exert their various and significant effects. Little is known about precisely how these

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orally-dosed polysaccharides act, yet understanding the mechanism is critical to both

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research and pharmaceutical product development.

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1. The concern on the quality standardization of polysaccharides.

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Currently, developing standards for the quality control of polysaccharides is hindered

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by one major difficulty: poor repeatability of the methods used for sample preparation

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and structure characterization. Take Ganoderma lucidum as example, which is one of

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the mostly studied mushroom. Although there are hundreds of research articles

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reporting the polysaccharides,4,5 none of them stated that their polysaccharides are

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identical to any previously reported. It is because that the repeatability of the

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separation method and the identification method is so poor that people are not able to

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confirm whether or not the polysaccharides they are studying is exactly same as any

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reported. 3



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The separation methods we are using decide that we are not able to isolate two

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completely identical polysaccharides from natural resources. Unlike small molecules,

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polysaccharides naturally exist as a mixture; a single polysaccharide molecule has

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never been obtained from natural resources. Separation and purification of a

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polysaccharide from food materials always involve a complicated procedure, in which

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water extraction, ethanol precipitation, deproteination might be the commonly used

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operation before repeated chromatographic purification. Take ethanol precipitation as

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an example. The response of polysaccharide precipitates to ethanol concentration

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varies greatly in terms of precipitate yield and molecule size, due to the diverse

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chemical structures of their components.6 Not to say that the ethanol concentration

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used here has never been fixed to an accurate value as 80.15% or 70.06%.

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Now chromatography has been popularly used in separation of polysaccharides

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because many new separation materials are adopted and the polysaccharide fractions

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could be greatly purified, similarly as we did to small molecules. However, the

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outcome of chromatographic fractionation is affected by many factors, while

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repeatability is one of the basic validation criteria for chromatography. Even the same

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crude polysaccharide sample, separated on the same separation material, in the same

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column, eluted with the same mobile phase and sample collection procedure, has been 4


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known to produce inconsistent results if the same operator combined the fractions

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slight differently. Because the isolates are still mixtures, and the composition of the

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mixtures varies and depends on the natural materials, extraction methods, column

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type/size/flowrate/detection, and the operators. As each fraction is a specific mixture,

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a new mixture will be generated once different fractions are combined together.

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When identical samples cannot be prepared reliably, research work cannot be repeated

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nor generalized, and industry cannot create standardized products.

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Even the sample preparation could be reliably repeated, the subsequent structure

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characterization involves more complicated operations and also has a bigger concern

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of poor repeatability in methodology. The results of molecular size analysis varied

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greatly between different analytical methods; even for the same HPGPC method,

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different analytical columns and different reference standard polymers also caused big

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difference. In some cases, the sugar composition was directly determined only by

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integrating peak area without any standard references. Methylation analysis has been

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popularly used to indicate the sugar linkage composition,7 but the repeatability is so

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poor that no one has exactly repeated the results of any study. The normal methylation

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analysis protocol mainly has seven steps: methylation plus dialysis, commonly

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performed twice (some need more); hydrolysis, reduction, and acetylation followed 5



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by GCMS determination,7 and all of these are variable. Assuming that every dialysis

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operation and chemical reaction generates an acceptable RSD of 5%, the final RSD

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could be as large as 40%. All these above-mentioned variations are enough to create

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different characterization results.

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Taken together, the poor repeatability of methods used in sample preparation and

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chemical characterization make the quality control of highly diverse polysaccharides a

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challenge and concern. This in turn greatly limits the research and development of

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polysaccharide-based products.

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Continuous efforts are being made to solve this QC problem. In the Chinese

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Pharmacopoeia, the characteristics of Dendrobium polysaccharides are described in

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terms of a monosaccharide profile: only mannose and glucose in a ratio of 2.8:1-8:1.8

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But this method fails to distinguish individual Dendrobium species since all of them

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present an identical monosaccharide profile. To overcome this failure, a holistic

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polysaccharide marker DOP has been successfully adopted in the qualitative and

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quantitative analysis of Dendrobii Officinalis Caulis.9 This polymer marker is unique

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and specific enough for qualitative authentication purposes, and the quantitation

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results are comparable to that of quantitative determination of the monosaccharide 6


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profile. However, this polysaccharide marker approach only fit a few cases since not

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all natural products contain such a special polymer marker,9 and it may not work for

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formula products. Oligosaccharides, with structures somewhere between monomers

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and polymers, offer another option for a unique marker. Oligosaccharide markers

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might contain both the bioactive structural unit and a specific structural feature.10,11

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Oligosaccharide mapping technology based on enzyme hydrolysis has been developed

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for authentication purposes.12-14

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2. The concern on the mechanism of action.

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In addition to the quality control issue, the lack of evidence regarding the mechanism

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of action is another major barrier to realizing the full potential of polysaccharides in

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the modern world. How polysaccharides work is a true puzzle.

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polysaccharides are ingested orally, either in decoctions or as pills or capsules.

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such, they are known to be poorly absorbed---yet they have important observable

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physiological effects. There are three theories as to their mechanism of action:

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absorption; via gut microbiota, or via Peyer’s patches.

Normally, As

direct

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Although almost all the current pharmacokinetic studies focused on the sample by i.v. 7



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administration, some scientists have made efforts to prove that polysaccharides are

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absorbed in the gastrointestinal tract. They have used various labeling reagents,

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especially fluorescent, to increase detection sensitivity;15-20 however, none of these

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fluorescence-labeling methods themselves have been well validated so results from

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using them may not be reliable. Did the polysaccharides, after being labeled, retain

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their original bioactivities and physical/chemical properties? Is it possible that the

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original polysaccharide was not absorbable but the labeling reagent changed the

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structure of the original polysaccharide such that it was then absorbed? Was the

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stability of the labeled polysaccharide determined? Did the labeled polysaccharide

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contain any free labeling reagent? Was the purity of the labeled polysaccharide

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checked using HPGPC? Without satisfactory validation of the analytical method, such

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absorption results are suspect.

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In addition to direct gut absorption, there are two other feasible mechanisms by which

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orally dosed polysaccharides can have significant impact. These are via gut

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microbiota or via Peyer’s patches. In the past ten years, research on gut microbiota

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has flourished, and the interaction between polysaccharides and gut microbiota has

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been a focus of many studies. Xu et al. have reviewed the literature regarding the role

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of polysaccharides as prebiotics.21 Xu et al. have reviewed the literature regarding the 8


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role of polysaccharides as prebiotics,21 and offer convincing evidence that

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polysaccharides work by altering the species composition of the gut microbiome.

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Here we would like to propose another possibility, Peyer’s patches. It is hypothesized

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that polysaccharides, without need to enter the blood/lymph circulation, trigger

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immune response immediately after entering Peyer’s patches where they activate

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innate immune cells. A recent study has indicated that after a three-hour journey in the

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small intestine, Angelica polysaccharide was quickly digested when it arrived in the

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large intestine.20 This means that the polysaccharide stays longer in the small intestine

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than anywhere else, and suggests that the small intestine is where it exerts its effects.

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Peyer’s patches, as the main immune organ in the small intestine, are small masses of

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lymphatic tissue found throughout the ileum. These patches contain several groups of

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immune cells such as T cells, dendritic cells, and macrophages, all of which are

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targets of polysaccharides.22-25 The question that next arises is, how do these

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polysaccharides enter Peyer’s patches? In the study of Rice et al,18 flow cytometry

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analysis of gut-associated lymphoid tissue (GALT) cells isolated from Peyer’s patches

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of mice gavaged with fluorescently labeled glucans, revealed that GALT cells are

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capable of recognizing and binding glucans. Uptake and internalization of

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fluorescently labeled glucans by murine gastrointestinal epithelial cells were also 9



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observed using confocal imaging. However, the epithelial cells have not been

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specifically identified. Once identified, as with gut microbiota, there will be complete

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evidence that Peyer’s patches are intimately involved in the biochemical mechanism

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by which polysaccharides exert their effects.

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Conclusion and suggestions.

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In summary, lack of quality control procedures for producing polysaccharides and

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lack of knowledge of the mechanism by which they work are delaying progress in the

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research and development of polysaccharide-based health products.

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to reach consensus on purity and structural characterization, and to determine reliable

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means to produce identical samples. As for mechanism, while some scientists pursue

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the possibility of direct gut absorption, increasing evidence points to the gut

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microbiome and/or Peyer’s patches for solving the mystery of polysaccharide activity.

Scientists need

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Correspondence should be addressed to

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*(Q.-B.H.) Mail: 7 Baptist University Road, School of Chinese Medicine, Hong Kong

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Baptist University, Kowloon Tong, Hong Kong SAR, China. Phone: 00852-34112906.

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Fax: 00852-34112461. E-mail: [email protected].

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Funding

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The author was financially supported by HKSAR Innovation and Technology Fund

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(ITF), Tier 3, ITS/311/09, General Research Fund (12100615, 22100014), Health

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Medical Research Fund (11122531, 14150521), National Natural Sciences Foundation

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in China (81473341), and Hong Kong Baptist University (RC-start up grant,

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MPCF-001-2014/2015, RC-IRMS/14-15/06, FRG1/16-17/032 and FRG2/16-17/002).

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References:

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[1] Schepetkin, I.A.; Quinn, M.T. Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Internat. Immunopharm. 2006, 6, 317−333. [2] Carbonero, E.R.; Gracher, A.H.P.; Komura, D.L.; Marcon, R.; Freitas, C.S.; Baggio, C.H.; Santos, A.R.S.; Torri, G.; Gorin, P.A.J.; Iacomini M. Lentinus edodes

206 207 208

heterogalactan: Antinociceptive and anti-inflammatory effects. Food. Chem. 2008, 111, 531−537. [3] Yang, L.Q.; Zhang, L.M. Chemical structural and chain conformational

209

characterization of some bioactive polysaccharides isolated from natural sources.

210 211 212 213 214 215 216

Carbohydr. Polym. 2009, 76, 349−361. [4] Ferreira, C.F.R.; Heleno, S.A.; Reis, F.S.; Stojkovic, D.; Sokovic, M. Chemical features of Ganoderma polysaccharides with antioxidant, antitumor and antimicrobial activities, Phytochemistry. 2015, 114, 38−55. [5] Shaoping Nie, Hui Zhang, Wenjuan Li, Mingyong Xie, Current development of polysaccharides from Ganoderma: Isolation, structure and bioactivities, Bioact. Carbohydr. Diet. Fibr. 2013, 1, 10−20.

11



217 218

[6] Xu, J.; Yue, R.J.; Liu, J.; Ho, H.M.; Yi, T.; Chen, H.B; Han, Q.B. Structural diversity requires individual optimization of ethanol concentration in precipitation of natural

219 220 221

polysaccharides. Internat. J. Biol. Macromol. 2014, 67, 205−209. [7] Filomena, A.P.; Cherie, W.; Geoffrey, B.F.; Antony, B. Determining the polysaccharide composition of plant cell walls, Nat. Protoc. 2012, 7, 1590−1607.

222 223

[8] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China. 2015, 1, 282−283.

224

[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

225 226

strategy for quality control of saccharide-dominant herbal materials: Dendrobium

227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243

officinale, 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. PNAS. 2015, 112, 8284−8289. [11]Li, S.P.; Wu, D.T.; Lv, G.P.; Zhao J. Carbohydrates analysis in herbalglycomics. Rev. 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.

244 245 246

[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. Receptor-mediated

247

absorption of high molecular weight dextrans from intestinal tract. Control. Rel.

248 249 250

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

251 252 253 254

and cellular immunity. Internat. 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 12


Page 12 of 14

Page 13 of 14


255 256

absorption of soluble glucans stimulate increased resistance to infectious challenge, J. Pharmacol. & Exper. Therap. 2005, 314, 1079−1086

257 258 259

[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,

260 261

820−825. [20]Wang, K.P.; Cheng, F.; Pan, X.L.; Zhou, T.; Liu, X.Q.; Zheng, Z.M.; Luo, L.; Zhang,

262 263 264 265 266

Y. Investigation of the transport and absorption of Angelica sinensis polysaccharide through gastrointestinal tract both in vitro and in vivo. Drug Deliv. 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,

267 268 269 270 271 272 273 274 275 276 277 278 279 280 281

1140−1185. [22]Zhang, J.; Tang, Q.; Zhou, C.; Jia, W.; Da Silva L; Nguyen LD; 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 sciences. 2010, 87, 628−637. [23]Kim, H.Y.; Kim, J.H.; Yang, S.B.; Hong, S.G.; Lee, S.A.; Hwang, S.J.; Shin KS; Suh HJ; Park MH. 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. 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. Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells. J. Pharm.

282

Pharmacol. 2011, 63, 725−735.

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Critical Problems Stalling Progress in Natural Bioactive

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