Critical problems stalling progress in natural bioactive polysaccharide

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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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Critical problems stalling progress in natural bioactive polysaccharide

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