A review of isolation methods, structure features and bioactivities of polysaccharides from Dendrobium species

Traditional Chinese medicine is the gem in China and many of them have been used for thousands of years for their multiple bioactivities. Among them, Dendrobium is a representative one which contains thousands of species. Most rare Orchidaceae species are endangered while have very high economic value. Its stems and flowers have been valued as precious food and herb medicine with healthy benefits and therapeutic effects of enhancing immunomodulation, promoting the body fluid production, inhibiting throat inflammation and curing fever, chronic superficial gastritis, and nurishing Yin. These beneficial activities have been attributed to complicated bioactive constituents of Dendrobium, while some chemical ingredients have been identified, including alkaloids ^[1-6], amino acid ^[7-9], trace elements ^[10-12], vitamins ^[13], polysaccharides ^[14-16] and other ingredients ^[17-20]. The amount of polysaccharide is an evaluation standard of Dendrobium quality in the Chinese pharmacopoeia (2015 Edition). Unlike proteins and nucleic acids, polysaccharides as another kind of important biomacromolecular can’t be sequenced while the determination of their exact structures and molecular size are still great challenges. As we know polysaccharides from various plants and fungus have gradually been identified and have many bioactivities with low or without cytotoxicity, including antitumor ^[21-22], immunomodulation ^[23-24], antimicrobial ^[25-26], antioxidation ^[27-28] and antifatigue ^[29-30]. Different polysaccharides from various Dendrobium species also have diverse activities and show complex structures which draw more attention as well as the relationships between chemical structures and bioactivities ^[31]. However, high medicinal value and limited resources make it expensive, which lead to the appearance of fake and inferior products. Thus, determination and elucidation of Dendrobium polysaccharides are significant for standardizing the market and further researches.

Recent decades, some of polysaccharides have been extracted from various Dendrobium species through different ways showing diverse structures and bioactivities. This review summarized recent advanced progress of extraction methods, structures, bioactivities of polysaccharides from Dendrobium.

In general, Dendrobium polysaccharides exist as a structural component of cell wall. So, which extraction method should be used depends on the cell wall structure. Besides, researchers aim to destroying the cell wall without damaging the structure of polysaccharides to make the extract as higher efficiency as possible. Dendrobium polysaccharides were extracted by solvent extraction methods, including hot water ^[32-33], alkali solution ^[34-35], acid ^[36] and polyethylene glycol (PEG) ^[37] after pretreatments, and combined with some supplementary methods to modify the extraction process and to obtain high yields, such as microwave ^[38], ultra-high pressure ^[39], ultrasonic ^[40-41], enzymatic ^[42-45]. As for the yield is concerned, the extraction temperature, pH of solvent, extraction time and ratio of material to solution are also crucial. The hot water extraction is still a popular and widely used approach. In brief, the hot water extraction ^[32-33] procedures are summarized as follows: dried Dendrobium stems were usually pretreated with ethanol or acetone to remove some small hydrophobic molecules and then crush into powder which was further extracted with hot water for several times monitored by phenol-sulfuric method. After concentrated and centrifuged, the supernatant was dialyzed against running water for 2 days. The internal solution was then centrifuged and precipitated by 80% ethanol overnight, then centrifuged and the residue was alternately washed by absolute ethyl alcohol and acetone respectively to yield the crude polysaccharide, and could be further deproteinized or not. In general, the crude polysaccharide containing starch ^[46] removed by applying amylase treatment, and further purified and fractionated by ion-exchange chromatography, gel filtration chromatography and affinity chromatography as well. In addition, alkali-extracted polysaccharide could be obtained with sodium hydroxide ^[35]. Dried stems of Dendrobium were soaked with 95% ethanol for two weeks, then air-dried and ground to obtain a fine powder. After the residue was extracted with boiling water, the filtered residue was further treated with 1 M sodium hydroxide in ice-cold bath water twice, 3 h for each time. Then alkali-extracted solution was neutralized and concentrated and dialyzed, followed by centrifugation and precipitation with 5 volumes of 95% ethanol. The precipitate was dried to obtain the crude alkali-extracted polysaccharide. As for the acid-extracted polysaccharide ^[47], it could be obtained with hydrogen chloride, the stems of Dendrobium were extracted for three times with hot distilled water, and then the residue was extracted with 5% HCl at 4 °C for 10 h to give acid extract polysaccharide. Polyethylene glycol (PEG) as a green solvent was employed to extract polysaccharide, and the extraction yield could increase with the help of ultrasonic ^[48-49]. The pretreated powder was extracted with PEG aqueous solution with the ultrasound device twice. The final extraction solution was centrifuged and precipitated with 5 volumes of 95% ethanol and the precipitate was dried to obtain crude polysaccharide. Furthermore, Ultra-high-pressure extraction technique was also employed to get higher yield of polysaccharides from Dendrobium with optimization of extraction pressure, pressure holding time and liquid-solid ratio ^[50]. Extraction with enzymes was mild, and could effectively destroy cell walls to release more polysaccharides, and this method is low carbon and environmentally friendly which is beneficial to industrialization.

Structures of polysaccharides from Dendrobium species always vary from each other, for their different sources, culture conditions, growing periods, plant parts, extraction methods and treatment processes (Table 1). Some homogeneous polysaccharides have been isolated and purified from several Dendrobium plants, and their structures and molecular weights also have been elucidated. Remarkably, Dendrobium plants from different sources owned their unique structures, and distinctions existed in not only backbones but also branches. However, the monosaccharide compositions and linkage styles are frequently similar to each other. In order to make the current research status clear, structures and some chemical parameters were summarized by species as follows:

Specie	Name	Mw (Da)	Sugar composition	Structural features	O-Acetyl groups	Ref.
D. huoshanense	DHP-4A	2.35 × 105	Glc : Man : Ara: Rha = 13.8 : 6.1 : 3.0 : 2.1		−	[51]
	DHP1A	6.7 × 103	Man : Glc : Gal = 2.5 : 16 : 1.0		Found but not assigned	[52]
	DHPD1	3.2 × 103	Gal : Glc : Ara = 0.021 : 1.023 : 0.023	(1→4)-linked-Glcp,(1→4,6)-linked-Glcp,(1→6)-linked-Galp, (1→6)-linked-Glcp, (1→5)-linked-Araf and T-inked-Glcp	−	[53]
	DHPD2	8.09 × 106	Gal : Glc : Ara = 0.896 : 0.723 : 0.2		−	[54]
	DHP-W2	7.3 × 104	Glc : Xyl : Gal = 1.0 : 1.0 : 0.5		−	[55]

Table 1.  Structure features of polysaccharides form Dendrobium species

Some water-extracted homogeneous polysaccharides with the molecular weights range from 3 × 10³−1 × 10⁷ Da have been analyzed in past years. They usually contained glucose, mannose and galactose. In addition, xylose, rhamnose, arabinose will randomly appear. Some of homogeneous polysaccharides were acetylated. Compared with other species, more homogeneous polysaccharides were fine characterized.

A homogeneous polysaccharide DHP-4A with molecular weight of 2.35 × 10⁵ Da was purified by DEAE-cellulose column and Sephacryal S-200 column from stems. GC-MS analysis indicated that DHP-4A was composed of glucose, mannose, arabinose and rhamnose in a molar ratio of 13.8 : 6.1 : 3.0 : 2.1. It contained a backbone of (1→6)-linked glucose, (1→6)-linked mannose and (1→3, 6)-linked mannose, with branches attached by β-L-Rhap-(1→2)-β-L-Rhap-(1→4)-β-D-Manp-(1→ and α-L-Araf-(1→3)-α-L-Araf-(1→3)-α-L-Araf-(1→substituted at the C-3 position of (1→6)-linked mannose ^[51].

A fraction (designated DHP1A) was isolated and the structure was elucidated by Tian et al ^[52]. This polysaccharide also contained mannose, glucose and a trace of galactose with a relatively low Mw of 6.7 × 10³ Da. According to the results of FT-IR, methylation analysis and NMR spectra, the backbone of DHP1A was demonstrated to be consisted of (1→4)-linked α-D-Glcp, (1→6)-linked α-D-Glcp, (1→4)-linked β-D-Manp, (1→4, 6)-linked α-D-Glcp which was substituted by the terminal β-D-Galp at O-6 position.

DHPD1 and DHPD2 were two homogeneous polysaccharides and their Mw (3.2 × 10³ Da and 8.09 × 10⁶ Da) were calculated and estimated by high-performance liquid chromatography (HPLC) based on the calibration curve ^[53-54], respectively. Monosaccharide analysis results indicated that both of them were mainly composed of galactose, glucose and arabinose while different in ratios (0.021 : 1.023 : 0.023 and 0.896 : 0.723 : 0.2, respectively) while trace of mannose and xylose existed in DHPD1. The glycosyl linkage styles of DHPD1 was partial elucidated that it contained (1→4)-linked-Glcp, (1→4, 6)-linked-Glcp, (1→6)-linked-Galp, (1→6)-linked-Glcp, (1→5)-linked-Araf and terminal linked-Glcp in the ratio of 7.23 : 1.5 : 0.21 : 0.18 : 0.26 : 1.24. In addition, a trace amount of terminal-linked-Xylp and (1→3, 6)-linked-Manp were also found based on results of methylation analysis, FT-IR and ¹³C NMR spectra. While the detail structure of DHPD2 which was isolated from protocorm liked bodies of D. huoshanense was successfully characterized. This polysaccharide was a long-chained hemicellulose with a few branches, according to the result of methylated hydrolyzates. Based on reference data and NMR spectroscopy, the results showed that DHPD2 had a backbone consisting of (1→5)-linked α-L-Araf, (1→6)-linked α-D-Glcp, (1→4) and (1→6)-linked β-D-Glcp, (1→6) and (1→3, 6) linked β-D-Galp with T-linked xylose and trace amount of T-linked mannose attached to O-3 position of (1→3, 6) linked β-D-Galp. In addition, according to the NMR spectra exhibited on references, signals belonging to the acetyl groups could be observed, which also need further identified. A water-soluble fraction (designated as DHP-W2) was isolated from fresh stems and purified with DEAE cellulose anion-exchange column and Sephacryl S-200 column. Its Mw was 7.3 × 10⁴ Da ^[55]. DHP-W2 was completely hydrolyzed by 2 mol·L^–1 trifluoroacetic acid and sugar composition was analyzed by GC-MS. The results revealed that it was mainly comprised of glucose, xylose and galactose in the molar ratio of near 1.0 : 1.0 : 0.5 and trace of galacturonic acid was observed as well. Based on the results of methylation analysis and NMR spectroscopy, it can be proposed that DHP-W2 had a backbone consisting of (1→4), (1→6) and (1→4, 6)-linked β-D-Glcp with branches of T-linked α-D-Galp and T-linked α-D-Galp attached to the O-6 of (1→4, 6)-linked β-D-Glcp, and (1→4), (1→2, 4) and T-linked α-D-Xylp, T-linked α-D-Galp and T-linked α-D-Galp attached to the O-4 of (1→4, 6)-linked β-D-Glcp directly or indirectly ^[55].

A galactoglucomannan (GGM, Glu : Man : Gal = 31 : 10 : 8) designated as HPS-1B23 was obtained from the stems with an average molecular weight of 2.2 × 10⁴ Da. HPS-1B23 contained a backbone of (1→6)-linked α-D-glucose with O-acetyl groups joined to the C-3 position, (1→4)-linked α-D-glucose and (1→3, 6)-linked α-D-mannose and branches of α-D-galactose in a molar ratio of 2.4 : 1 : 1 : 0.9 ^[56]. A fraction B that was a partial acetylated glucomannan of ~60 DP (~1 × 10⁴ Da) was isolated from the mucilage of leaves and stems from D. huoshanense. Combined with chemical and enzymatic methods, 1D and 2D NMR spectra, monosaccharide composition (Man : Glc/10 : 1), the tentative partial structure of this sugar was determined. The results suggested that fraction B had a backbone consisting of (1→4)-linked Glcp and Manp, and part of (1→4)-linked Manp was mono-substituted by acetyl groups at position O-2 or O-3, while no branches were found ^[57].

A homogenous polysaccharide (GXG) ^[58] was obtained from fresh stems of Dendrobium huoshanense, and the molecular weight of this polysaccharide was 1.78 × 10⁶ Da. Monosaccharide compositions analysis result showed that GXG was composed of xylose, galactose and glucose in a molar ratio of 2.13 : 1.00 : 2.85. There were nine different glycosyl residues including terminal-linked Xylp, (1→4)-linked Xylp, (1→2, 4)-linked Xylp, terminal-linked Galp, (1→4)-linked Galp, (1→3, 6)-linked Galp, (1→6)-linked Glcp, (1→4)- linked Glcp and (1→4, 6)-linked Glcp existed in GXG.

A homogenous glucomannan cDHP ^[59] was got from dried D. huoshanense stem. cDHP was mainly composed of mannose and glucose in a molar ratio of 1.89: 1.00, and had a backbone with linkages of (1→4)-Manp, (1→4)-Glcp, (1→4, 6)-Manp and Terminal-Glcp. From the IR spectrum, the presence of acetyl groups at 1732 and 1637 cm^–1 suggested that the cDHP was an acetylated polysaccharide, which contained 4.57% ± 0.96% O-acetyl groups based on the determination of back-titration method.

Notably, Tie-pi-shi-hu growth in China is designated as Dendrobium officinale but not Dendrobium candidum. In this paper, Dendrobium candidum was corrected as Dendrobium officinale ^[60].

So far, polysaccharides from D. officinale were always detected as glucomannan or mannoglucan with (1→4)-linked glucose or/and mannose in a β-configuration acetylated in varying degrees and positions with or without branches. But only two structures were fine elucidated, because the existence of acetyl groups made it hard to be characterized.

The structure and properties of a 2-O-acetylglucomannan (designated DOP-1-A1) was elucidated by Hua et al ^[61]. It was composed of mannose, glucose and arabinose with the molar ratios of 40.2 : 8.4 : 1 and HPGPC result showed that it was a homogeneous polysaccharide and its molecular weight was 1.3 × 10⁵ Da. The backbone and branches were determined by methylation analysis and monosaccharide analysis of both native and partially hydrolyzed polysaccharides. The result indicated that the backbone consisted of (1→4)-linked β-D-Manp and (1→4)-linked β-D-Glcp, while branches were suggested to join in O-6 position to which terminal-linked and (1→3)-linked β-D-Manp and β-D-Glcp, a small number of T-linked Araf residues attached. Furthermore, combined the results of methylation analysis, periodate oxidation, chemical method of deacetylation by the strongly alkaline condition with NMR spectra, the acetyl groups were found attached to the O-2 position of (1→4)-linked β-D-Manp and (1→4)- linked β-D-Glcp.

Later, another acetylated linear glucomannan (designated DOP) was isolated with a relative larger Mw (3.12 × 10⁵ Da), while it mainly contained mannose and glucose in a molar ratio of 6.9 : 1. Compared with the DOP-1-A1, DOP possessed the same backbone without branches, while the position of acetyl groups was only attached to O-2 or O-3 position of mannose based on the results analyzed by 1D NMR, 2D TOCSY and COSY spectra ^[62]. However, the exact structure was difficult to elucidate for the relatively high viscosity. To obtain more detail structural information, researchers used endo-β-mannanase to produce a relatively low viscosity oligosaccharide (designated HDOP) rich in O-acetyl groups. Finally, high-resolution heteronuclear 2D NMR spectra was obtained. Combined with 1D NMR, 2D COSY, 2D NOESY, 2D TOCSY, 2D HMQC and 2D HMBC spectra, the position of acetyl groups was further identified. Besides the O-2 or O-3 position of some mannosyl residues was acetylated, there were small amount of it with di-O-acetyl substitutions at both the O-2 and O-3 positions. Moreover, minor levels of 6-O-acetylated and 2, 6-di-O-acetyled mannose residues were found. In addition, during the process of structure analysis, researchers found that 2-O-acetyl and 3-O-acetyl substitutions of mannose residues could change the ¹H chemical shift of its neighbouring residues at the reducing end side, which were defined as “M2 effect” and “M3 effect” ^[63]. Moreover, another partial elucidated neutral polysaccharide DOP-1-1 which contained acetyl groups as well based on the results of FT-IR and NMR spectra was also composed of mannose and glucose in a molar ratio of 5.9 : 1 with an average molecular weight of 1.78 × 10⁵ Da ^[64]. Its sugar composition was similar to DOP. However, more information about its structure still needed to be characterized. A new homogeneous polysaccharide (DOP-W3-b) with a relatively low molecular weight of 1.54 × 10⁴ Da was obtained through a bioactivity-guided sequential isolation procedure ^[65]. DOP-W3-b had a backbone consisting of (1→4)-linked β-D-Manp, (1→4)-linked β-D-Glcp and (1→3, 6)-linked β-D-Manp residues and branches consisting of (1→4)-linked β-D-Glcp, (1→4)-linked β-D-Manp and terminal β-D-Glcp, with O-acetyl groups attached to O-2 of (1→4)-linked β-D-Manp only. However, the fine structure still needs to be elucidated, such as the proportion of (1→4)-linked β-D-Manp and (1→4)-linked β-D-Glcp in branched chain and main chain, the positions that side chains attached to and the degree of acetylation. In addition, there was new glucomannan named DOP ^[66]as well was obtained and molecular weight of it was 3.95 × 10⁵ Da, it was mainly composed of two monosaccharides including mannose and glucose, with a ratio of 3.8 : 1.0.

A primary research characterized two homogeneous linear glucomannans DOPA-1 and DOPA-2 with average molecular weights of 3.94 × 10⁵ and 3.62 × 10⁵ Da, respectively. Both polysaccharides were mainly composed of mannose and glucose ^[67]. Not surprisingly, they contain a backbone consisting of (1→4)-linked β-D-Manp and β-D-Glcp with O-acetyl groups. However, information about side chain of these two polysaccharides was not provided.

Besides, DOP-1 (5.34 × 10⁵ Da) and DOP-2 (1.60 × 10⁵ Da) were found mainly composed of mannose, glucose, a trace amount of galactose and arabinose and the molar ratios were similar to each other. However, more data still need be required for their precise structural analysis ^[34].

There were other homogeneous polysaccharides were purified designated DOPW-1 (7.8 × 10⁴ Da), DOPW-2 (3.7 × 10⁴ Da), DOPS1-1 (2.87 × 10⁵ Da), DOPS1-2 (3.51 × 10⁵ Da), DOPS1-3 (3.35 × 10⁵ Da) and DOPS1-4 (1.71 × 10⁵ Da) ^[68]. Primary structure analysis found that all of them mainly composed of galactose, arabinose and glucose residues. However, their structures are not elucidated yet. However, another group isolated two novel polysaccharides designed as DOPW-1 and DOPW-2 as well. Their molecular weights were 389.98 and 374.11 kDa, respectively ^[69]. Both the two polysaccharides were glucomannan. DOPW-1 consisted of mannose and glucose at a ratio of 10.75 : 1, whereas DOPW-2 consisted of mannose and glucose at a ratio of 8.82 : 1. Methylation results showed that both DOPW-1 and DOPW-2 had the same backbones of →4)-β-D-Manp-(1→ and →4)-β-D-Glcp-(1→, albeit with different percent contents, with their branched chains consisting of terminal Manp at O-3 and O-6. The acetyl groups were attached to the O-2 of mannose residues in the main chain.

DCPP3c-1 and DCPP1a-1 were two homogeneous fractions with average molecular weight of 7.24 × 10⁴ and 1.89 × 10⁵ Da, respectively ^{[70- 71]}. DCPP3c-1 mainly contained galacturonic acid, while DCPP1a-1 was a glucomannan. Both of them were composed of (1→6)-linked, (1→2)/(1→4)- linked and (1→3)-linked glycosidic bonds in different molar ratios.

A homogenous polysaccharide designed S32 ^[35] was obtained and the molecular weight was 3.7 × 10⁴ Da. Monosaccharide composition analysis results showed that it contained arabinose, xylose, glucose and 4-O-methylglucuronic acid (4-MGA) as well as trace amount of rhamnose and galactose in a molar ratio of 8.9 : 62.7 : 8.5 : 12.3 : 3.9 : 3.7. Combining with the results of monosaccharide composition, methylation analysis and NMR spectrum, this polysaccharide contained a backbone of (1→4)-linked-β-D-xylan, with branches of (1→4)-linked-α-D-glucose, (1→3)-linked-α-L-rhamnose, and terminal-linked-α-L-arabinose, β-D-galactose, 4-MGA, and β-D-xylose directly or indirectly attached to C-2 of glycosyl residues on backbone.

Furthermore, a heteropolysaccharide designed DOP-GY ^[72] extracted from the stems of D. officinale was also purified by ion-exchange chromatography on DEAE and Sephadex G-200 column. The molecular weight of this polysaccharide was 9.52 × 10⁵ Da. Pre-column derivatization HPLC was employed to analysis the monosaccharide composition and the result showed that it was composed of glucose, galactose and mannose in the molar ratio of 2.13 : 1.34 : 1.00. However, the detailed linkage styles and structure feature of this polysaccharide was not characterized.

To date, some polysaccharides from D. nobile Lindle had been elucidated. However, their monosaccharides and structures were multiple. Wang et al had achieved water-extracted (DNP-W), 5% NaOH-extracted (DNP-OH) and 5% HCl extracted (DNP-H) crude polysaccharides. Then the DNP-W was further purified by anion-exchange chromatography eluted with water in a stepwise way by 0.05, 0.1, 0.2, 0.3 and 0.5 mol·L^–1 NaCl solutions and six kinds of polysaccharides had been obtained (designated DNP-W1 to DNP-W6) ^[36]. Some of those six sub-fractions had been further purified and their structural features had been described. Crude polysaccharide DNP-W was firstly fractionated by DEAE-cellulose anion-exchange chromatography, and a fraction eluted by 0.05 mol·L^–1 NaCl was further purified by gel-chromatography to obtained DNP-W2. Unlike other polysaccharides obtained from D. nobile Lindl., DNP-W2 (Mw: 1.8 × 10⁴ Da) was acetylated at the O-2 position of (1→4)-linked β-D-Manp ^[73]. It was composed of mannose, glucose and galactose in a molar ratio of 2.9 : 6.1 : 2.0. Its backbone and branches were analyzed and characterized by partial acid hydrolysis and methylation. It contained a backbone of (1→6)-linked β-D-Glcp, (1→4)-linked β-D-Glcp and β-D-Manp, with terminal-linked α-D-Galp attached to the O-6 position of (1→4)-linked β-D-Glcp and β-D-Manp. The structure features are similar to that of D. officinale. Nevertheless, the fine structure need be elucidated combining with other instruments and chemical methods.

DNP-W5 was a RG-II polysaccharide which was composed of Rha, GalA, Man, Gal and Xyl in a molar ratio of 4.2 : 3.9 : 3.1 : 8.1 : 8.2 : 0.6 ^[74]. It contained a similar backbone of RG-Ⅱ with (1→4)-linked β-D-Glcp, (1→6)-linked β-D-Manp and terminal-linked β-D-Galp attached to O-4 of the Rhap, and Xylp residues substituted at O-3 of (1→3, 4)-linked Galp. What’s more, 6.9% O-acetyl groups could also be examined. 2D NMR spectra should be employed to identify the fine structure.

DNP-W3 was a kind of rhamnoarabinogalactan with its Mw of 7.1 × 10⁵ Da, which eluted by 0.1 mol·L^–1 NaCl on DEAE-Cellulose and purified by Sephacryl S-200. Monosaccharide analysis indicated that it was comprised of Gal, Rha and Ara in a molar ratio of 3.1 : 1.1 : 1.0 ^[75]. Structural analysis indicated that it contained a backbone of (1→3)-linked β-D-Galp and branches which were (1→4)-linked α-L-Rhap and T-linked β-L-Arap attached to the O-4 position of main chain occasionally.

In addition, a homogeneous polysaccharide DNP (8.76 × 10⁴ Da) was prepared. It composed of Rha, Ara, Xyl, Man, Glu and Gal in a molar ratio of 1.00 : 2.80 : 2.20 : 30.76 : 117.96 : 31.76. This polysaccharide had a backbone of (1→6)-α-D-Glcp, (1→6)-α-D-Galp with terminal-linked α-D-Manp attached to C-4 position of (1→6)-α-D-Galp and terminal-linked α-D-Glcp attached to C-4 position of (1→6)-α-D-Glcp. Noticeably, strong signals not assigned around δ 2.0 ppm in ¹H NMR spectrum and δ 21 ppm in ¹³C NMR spectrum might originate form O-acetyl group ^[76].

JCP-40 ^[77] was a homogeneous water-soluble neutral glucomannan obtained from Dendrobium nobile Lindle. The average molecular weight of this polysaccharide was 6.75 × 10⁴ Da. Monosaccharide composition analysis showed that JCP-40 consisted of mannose (74.97 wt%) and glucose (25.03 wt%). The structural features of JCP-40 was proposed according to monosaccharide composition analysis, methylation analysis, 1D/2D NMR spectroscopy. JCP-40 was a linear polysaccharide which was consisted of →4)-β-D-Manp-(1→ and →4)-β-D-Glcp-(1→. The content of O-acetyl group on JCP-40 was approximately 1.6%, and acetyl groups were attached to the C-2 or C-3 on mannosyl residues based on NMR spectrum results.

DNP1-1, DNP2-1, DNP3-1 and DNP4-2 were four homogeneous fractions obtained from D. Nobile Lindle with the average molecular weight of 1.36 × 10⁵, 2.77 × 10⁴, 1.18 × 10⁴ and 1.14 × 10⁴ Da, respectively. Monosaccharide composition analysis indicated that the four polysaccharides consisted of similar glycosyl residues. All of them mainly contain mannose, glucose, and galactose ^[78].

A water-soluble polysaccharide JCS1 ^[79] obtained from the stems of Dendrobium nobile with the molecular weight of 1.2 × 10⁴ Da. This polysaccharide was composed of glucose, mannose, xylose, and arabinose in a molar ratio of 40.2 : 2.3 : 1.7 : 1.0. Structural features of JCS1 were investigated by a combination of chemical and instrumental analysis. The results showed that JCS1 was a mannoglucan with a backbone consisting of (1→4)-linked-β-Manp and (1→4)-linked-α-Glcp attached by branches at C-6 of (1→4)-linked-α-Glcp residues. The branches were composed of terminal-α-Glcp, (1→4)-α-Xylp, and terminal-α-Araf.

Very few studies focused on structures of polysaccharides from Dendrobium densiflorum, instead, researches pay more attention on species discrimination and identification ^[80-84] as well regeneration ^[85].

So far, a homogeneous mannoglucan (DDP-1-D) was isolated and purified from D. densiflorum with an average molecular weight of 9.4 × 10³ Da ^[86]. Monosaccharide composition analysis indicated that DDP-1-D was also composed of mannose and glucose in a molar ratio of 1 : 3.01. Linkage patterns were identified by results of periodate oxidation-Smith degradation and FT-IR, 1D and 2D NMR spectra, which suggested that DDP-1-D has a main chain consisting of (1→4)-linked α-D-Glcp and (1→6)-linked α-D-Glcp, (1→2)-linked α-D-Manp and (1→4)-linked β-D-Manp. In addition, trace amount of (1→3)-linked glycosyl was observed, however no structural information about side chain was provided. Hence, more investigation could be performed to draw the conclusion.

To date, researches more concentrated on micromolecules ^{[87- 88]} and bioactivities of crude polysaccharides from this plant ^[89-90]. However, three water-soluble polysaccharides had been purified from crude polysaccharide designated DDP1-1, DDP2-1 and DDP3-1 with the average molecular weight of 5.15 × 10⁴, 2.61 × 10⁴ and 6.9 × 10³ Da, respectively. Like polysaccharides from other Dendrobium species, monosaccharide analysis suggested that DDP1-1 mainly contained glucose and mannose, while arabinose, xylose and galactose also existed in molar ratios of 140.82 : 57.11 : 1.00 : 2.82 : 7.76. DDP2-1 showed the same composition but different in molar ratios that was 77.5 : 1.18 : 1.00 : 1.62 : 7.79. Nevertheless, xylose was not found in DDP3-1which was consisted of glucose, galactose, mannose and arabinose in a molar ratio of 8.84:2.00:1.03:1.0 ^[91-92]. However, the homogeneity and structures of polysaccharides still need to be analyzed.

DDP was achieved with Mw of 4.85 × 10⁵ Da and was consisted of glucose, galactose, mannose, xylose and arabinose in a molar ratio of 34.20 : 10.16 : 8.92 : 2.66 : 1.00. Based on the Infrared spectrum, ¹H and ¹³C NMR spectra of DDP, some sugar residues were identified including T-linked α-D-Galp and α-D-Manp, (1→4, 6)-linked and (1→4)-linked α-D-Glcp ^[93]. However, the main chain and the side chain still need to be elucidated.

A homogeneous polysaccharide was isolated from Dendrobium tosaense designated DTP-N. It was composed of mannose, glucose and galactose in a molar ratio of 150.7 : 9.1 : 1 with molecular weight of 2.20 × 10⁵ Da ^[94]. Methylation analysis indicated that (1→4)-linked mannose was main linkage type of its backbone and other linkages also existed in main or side chain, including (1→3)-linked mannose and glucose, (1→6)-linked and (1→3, 4)-linked mannose, terminal linked glucose and mannose in a molar ratio of 59.42 : 3.73 : 3.9 : 11.61 : 16.12 : 0.54 : 4.68. Methylation analysis still need to be confirmed for that the molar ration of glycosyl residues of branch was not comparatively equal to that of terminal linked residues. Even though ¹H NMR was obtained, information stills not enough to fine elucidate the exact structure.

Eight kinds of homogeneous polysaccharides had been isolated from Dendrobium moniliforme by Chen et al ^[95], designated DMP1a-1, DMP1a-2, DMP2a-1, DMP3a-1, DMP4a-1, DMP5a-1, DMP6a-1 and DMP7a-1, some of which were characterized. Most of those polysaccharides showed the similar monosaccharide compositions with that of other Dendrobium species.

DMP1a-1 was mainly made up of mannose and glucose in a molar ratio of 4.798 : 1. Its molecular weight was 2.8 × 10⁴ Da. Periodate oxidation result indicated that β-(1→3)-linked, β-(1→4)-linked and β-(1→6)-linked glycosidic bond were found in a molar ratio of 23.59 : 8.75:1. Acetyl groups might exist based on the IR spectrum analysis.

DMP2a-1 was prepared from water extracted crude polysaccharide with DEAE-cellulose and gel-filtration chromatography. DMP2a-1 with molecular weight of 6.0 kDa was composed of glucose and mannose (12.6 : 1) as well as a trace amount of galactose. Combined with methylation and ¹H NMR and ¹³C NMR spectra analysis, the structure was proposed that it contained a backbone of (1→4)-linked α-D-Glcp, one sixth of which was substituted by branches (terminal-linked mannose and glucose) at the O-6 position ^[96].

Another homogeneous polysaccharide (3.05 × 10³ Da) fraction designated DMP4a-1 was also isolated. It showed more complex monosaccharide composition than that of DMP1a-1 and DMP2a-1 ^[97]. This polysaccharide was consisted of glucose, mannose, rhamnose, arabinose and galactose in molar ratios of 2.873 : 2.850 : 1.762 : 1.279 : 1.00. Periodate oxidation was employed to analyze the linkage styles and the result indicated that (1→3)-linked, (1→4)-linked and (1→6)-linked glycosidic bonds existed in molar ratios of 2.430 : 1.194:1.

One polysaccharide extracted from Dendrobium devonianum was designed as DDP, the molecular weight of DDP was 3.99 × 10⁵ Da ^[98]. Monosaccharide composition analysis showed that DDP was consisted of Man and Glc with the ratio of 29.61 : 1.00. Methylation analysis showed that the main linkage types in DDP were 1, 4-Manp and with trace of 1, 4-Glcp in the ratio of 27.21 : 1.00, and there was minor amount of T-Glcp. The degree of acetylated mannose was estimated to be 18.8%. Combined with NMR spectra, the DDP might had a backbone consisting of 1, 4-linked mannose, with minor 1, 4-linked glucose and free of branching. Acetyl groups were found attached to the O-2 or O-3 positions on some mannosyl residues.

Another novel polysaccharide obtained from D. devonianum, which was designated as DvP-1 with molecular weights of 9.52 × 10⁴ Da ^[99]. DvP-1 was a homogeneous heteropolysaccharide consisting of D-mannose and D-glucose in the molar ration of 10.11 : 1. Methylation result suggested that the main glycosidic linkages were β-1, 4-Manp, which were substituted with acetyl groups at the O-2, O-3 and/or O-6 positions.

Natural and modified polysaccharides usually showed multiple bioactivities. In recent years, pharmacological activity research of Dendrobium species indicated that polysaccharides usually exhibited antioxidant and immuno-enhancing activities. However, more and more evidence indicated that polysaccharides from Dendrobium species had multiple bioactivities including anti-tumor, hepatoprotective effect, anti-glycation and etc (Table 2). Noticeably, a lot of bioactivities testes mainly based on crude polysaccharide, which would make it hard to prove the exactly active substances. The activities of polysaccharides were summarized as follows:

With the advance of scientific research, people gradually realized that many diseases were related to the oxidation, and antioxidant strategy had attracted more and more attention. Polysaccharides from Dendrobium usually showed good antioxidant effect by reducing the production of free radicals, increasing the free radicals scavenging abilities, reducing protein decomposition, enhancing the capacity of antioxidant enzymes and so on.

Antioxidant properties in vivo and in vitro were tested by various different systems. To study the anti-oxidant activity of polysaccharides from Dendrobium species in vitro, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) test, 2, 2-azinobis-6-(3-ethylbenzothiazoline sulfonic acid) (ABTS) radicals scavenging assay, hydroxyl radicals scavenging assay were employed. Antioxidant properties in vivo were measured with different methods. The antioxidant enzymes, such as superoxidase dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), are remarkable molecules involved in antioxidation in vivo and also critical to evaluate the antioxidant capability in vivo. Malondialdehyde (MDA) as the end product of lipid peroxidation is a critical molecular in the formation of lipid radicals. Therefore, the amount of MDA in vivo can reflex the degree of lipid peroxidant to some extent.

DDP was a water-soluble polysaccharide extracted from Dendrobium denneanum ^[93]. In the experiments of DPPH radicals and hydroxyl radicals scavenging assay, its antioxidant activity increased with the increasing concentration and the antioxidant effects were 92.06% and 79.8% which were similar to that of vitamin C (P < 0.05) and even higher than of vitamin C in hydroxyl radicals scavenging assay at high concentration (2 mg·mL^–1). However, DDP had no significant scavenging effect on ABTS radicals. What’s more, antioxidant activity in vivo indicated that DDP could enhance the activity of SOD and inhibit the release of MDA significantly at the concentration of 200 mg·kg^–1.

The antioxidant activities of purified polysaccharides designated DDP1-1, DDP2-1 and DDP3-1 from D. denneanum were measured and the results showed that DDP2-1 exhibited strong antioxidant activity (89.3%) on DPPH radical scavenging assay especially in high dose (2 mg·mL^–1) as well as hydroxyl radicals scavenging assay (78.9%) with the same dosage in a dose-dependent manner. Both the DDP1-1 and DDP2-1 had very low ABTS and hydroxyl radical-scavenging power ^[92]. However, more in vivo investigations should be carried out.

Antioxidant activity in vitro of five water-soluble polysaccharide fractions (designated DNP, DNP1-1, DNP2-1, DNP3-1 and DNP4-2) purified from crude polysaccharide of Dendrobium nobile Lindl was investigated as well. DNP demonstrated very strong scavenging effect on ABTS radicals (78%) that was approximate to positive control (vitamin C) at 2 mg·mL^–1 and demonstrated an appreciable scavenging effect on hydroxyl radicals in dose-dependent manner. While it exhibited very low antioxidant activity on DPPH test ^[76]. With the same methods, the same group tested the antioxidant activity of another four kind of polysaccharides with vitamin C as positive control ^[78]. DNP4-2 demonstrated strongest scavenging activity on ABTS test and the hydroxyl radical test than that of all the others (P < 0.05) but weaker than that of control. However, even DNP4-2 showed stronger impact than that of others. But the scavenging effect on DPPH was very weak, with 38% scavenging effect at the concentration of 1.0 mg·mL^–1, while vitamin C was 95% at the same concentration.

Three polysaccharides (designated DO, p-DOP and c-DOP) were prepared from Dendrobium officinale and their antioxidant activities were investigated with cyclophosphamide induced immunosuppressed mice as model ^[100]. Huang et al evaluated the activities of antioxidant enzymes (SOD, CAT, GSH-Px), the total antioxidant capability (T-AOC) and the MDA levels in the serum, thymus and liver as well as effects on thymus and liver indices. The results showed that all of the three polysaccharides could enhance the activities of SOD, CAT and GSH-Px, and decrease the production of MDA in the liver, thymus and serum. The results accorded with that all the polysaccharides could increase the thymus and liver indices. The antioxidant effects possibly related to the existence of the O-acetyl groups and the β-(1→4)-linked mannose ^[57]. Interestingly, another water-extracted 2-O-acetylglucomannan with (1→4)-linked β-D mannose and glucose residues from D. officinale also had the antioxidant effect. The results indicated that DOP had good scavenging activity of DPPH radicals and hydroxyl radical ^[101]. DCPP and DCPP3c-1 were two polysaccharides isolated from suspension-cultured protocorms of D. officinale ^[102]. Both of them demonstrated strong scavenging ability on OH^– and O₂^–, and could inhibit the peroxidation of lipid induced by H₂O₂ and the production of MDA of mice liver as well. Moreover, hot water-extracted crude polysaccharide from D. fimhriatum Hook. var. oculatum (designated DFHP) showed excellent scavenging power on hydroxyl radicals at the concentrations ranging from 1.0 to 3.0 mg·mL^–1, while scavenging power on DPPH radicals was not as good as that on hydroxyl radicals even at high concentration (3.0 mg·mL^–1) ^[103]. Otherwise, D. fimbriatum polysaccharide collected from different regions exhibited different antioxidant activity ^[104].

Polysaccharides from Dendrobium species exhibited excellent immunoregulatory activities, especially those from the D. huoshanense, Dendrobium officinale and Dendrobium nobile Lindl. Immune activity tests indicated that those crude or purified polysaccharides usually could enhance the function of immune cells, increase the secretion of cytokines, and activate macrophages, etc.

Crude polysaccharide HPS and its sub-fractions were prepared and their immunomodulating activities in vitro were examined through measuring the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) of murine peritoneal macrophages and splenocytes ^[105]. The expression of corresponding cytokine gene was determined by RT-PCR. The results demonstrated that HPS could significantly enhance the release of TFN-α at the concentration of 200 μg·mL^–1 at the 24^th hour of culture and the amount reached to 542.9 pg·mL^–1 which was 1.5 times of LPS group induced, while the production start to decrease at concentration ranging from 400 to 800 μg·mL^–1. The change of TNF-α production at different time periods was in line with the expression of TNF-α gene. What’s more, the release of IFN-γ of splenocyte was also increased after HPS treated. The highest value was observed at the concentration of 800 μg·mL^–1 which was higher than that of the control. It was also found that its sub-fractions also could promote the release of IFN-γ and TNF-α at lower concentrations.

In another research, male Kunming mice were orally administrated with a galactoglucomannan purified from D. huoshanense (designated DHP) for 15 days. Results suggested that DHP could not only increase the proliferation of hepatocyte and splenocyte and promote the secretion of IFN-γ in the spleen, and IFN-γ and IL-4 in the liver, but also enhance the small intestinal immune and recovery its immune function suppressed by methotrexate ^[106]. It was proved that the acetyl group is necessary for the bioactivities. Another DHP polysaccharide purified from D. huoshanense by another group could effectively activate macrophages to release NO, TNF-α and IL-1β ^[107]. Experiments on RAW264.7 macrophages and peritoneal macrophages of C3H/HeN and C3H/HeJ suggested that toll-like receptor 4 (TLR4) was the essential receptor for DHP direct binding to macrophages and DHP activated cells via TLR4-mediated NF-κB, MAPKs and PI3K/Akt signaling pathways.

DH-PS, a crude polysaccharide from D. huoshanense, demonstrated the ability to promote the secretions of multiple cytokines and chemokines of BALB/c mice in vivo and as well as in human immune cells. Besides, DH-PS could significantly increase the production of IL-1ra both in mice and human monocytes (CD14⁺ and THP-1 cells). In addition, various kinases were investigated to find intracellular signaling involved in IL-1ra expression induced by DH-PS, while the results showed that ERK/ELK, p38 MAPK, PI3K and NF-κB were the signaling pathways involved in the expression of IL-1ra ^[108].

The crude polysaccharide DOP extracted from D. officinale and its homogeneous sub-fractions DOP-1 and DOP-2 exhibited the abilities to stimulate splenocyte proliferation and secrete cytokines IL-2 and IL-4, to activate macrophages to produce NO and cytokines TNF-α and IL-1β. These enhanced the phagocytosis of RAW267.4 cells significantly and cytotoxicity of natural killer (NK) cell ^[34]. DOPA, DOPA-1 and DOPA-2 polysaccharides from D. officinale were also found to show mild immunostimulatory and protect RAW 264.7 macrophages via promoting cell viability, ameliorating oxidative injury and suppressing apoptosis ^[67]. Polysaccharides obtained from D. officinale were usually elucidated as O-acetyl-glucomannan and the acetyl group was regarded as bioactive groups. However, more studies about the structure and bioactivity relationships and in vivo investigations using animals were needed. Another research team focused on immunomodulatory activity in vitro of crude polysaccharide (CDOP) and purified O-acetyl-glucomannan (PDOP) ^[109]. More deeply tests were carried out and researchers not only found that both of them could enhance the immunomodulatory activity through activating macrophages, upregulating cytokines secretion in vitro and stimulating production of NO as well, but also found the expression of inducible nitric oxide synthase (iNOS), TNF-α and IL-6 mRNA might be involved in immune response in early stage. Another O-acetyl glucomannan DOP-1-1 showed the immunomodulation activity might via activating the secretion of cytokines (IL-1β and TNF-α). The mechanism study results about involved intracellular signal passway indicated that DOP-1-1 could upregulate the phosphorylation of NF-κB and Erk1/2 and also inhibit the phosphorylation of Erk1/2 in THP-1 ^[64]. More interestingly, polysaccharides from the same species, the immune function of BALB/c mice was enhanced after being orally administered ^[110]. O-acetylated DOP-W3-b also displayed immunomodulating activities of intestinal mucosal and promoted the secretions of cytokines and increasing the production of secretory immunoglobulin A (sIgA) in the lamina propria ^[65]. O-acetylated polysaccharides could enhance the immune function through stimulating the secretion of cytokines splenocytes and/or macrophages, and/or upregulating T- and/or B-lymphocytes ^{[56-57, 73-74]}. Very interestingly, the deacetylated polysaccharide lost the aforementioned activity ^[74]. Moreover, crude polysaccharide (designated c-DOP) and its highly purified homogeneous O-acetyl glucomannan fraction (designated p-DOP) could attenuate immunosuppression in vivo induced by cyclophosphamide (Cy) in a dose-dependent manner. However, the p-DOP fraction treatment group showed stronger immunomodulatory activity, which indicated that the homogeneous polysaccharide was the pivotal bioactive component ^[111]. This confirms that the importance of the existence of acetyl group for immunological bioactivity.

Two homogenous polysaccharides DOPW-1 and DOPW-2 obtained from D. officinale, exhibited good immunomodulatory activity via promoting macrophage proliferation and increasing macrophage phagocytosis and nitric oxide production ^[69]. Fluorescence microscopy study of fluorescein isothiocyanate (FITC)-labelled DOPW-1 suggested that polysaccharide might attach to the surface of RAW 264.7 cells or enter the inside of the cells. These results indicated the significance of exploiting DOPW-1 and DOPW-2 not only as novel potential immunomodulators but also as beneficial tools focusing on studying the molecular mechanisms underlying polysaccharide efficacy.

Crude polysaccharides extracted with different methods and collected from different species showed different bioactivities. Enzyme-assisted extracted polysaccharide (designated DCP-E) showed higher capacity on splenic cell proliferation than that of hot water extracted crude polysaccharides (designated DCP-H) isolated from D. chrysotoxum ^[43]. The results might indicate that extraction efficiency by hot water is lower. Researchers compared the effects on macrophage of D. officinale from Yunnan, Anhui and Zhejiang, D. fimbriatum from Yunnan, D. huoshanense from Anhui, D. nobile and D. chrysotoxum from Yunnan, the results showed that crude polysaccharide from D. officinale of Yunnan exerted strongest activities in promoting phagocytosis, enhancing the release of some cytokines (TNF-α, IL-6, IL-10 and IL-1α) and NO ^[112]. Other researches in vitro and in vivo focused on polysaccharides from Dendrobium also indicated that they could induce the immunity activities to some extent ^[113-115]. Otherwise, the selenylation modification of polysaccharides could significantly enhance the immune activity through promoting lymphocytes proliferation ^[14].

Recent years, researchers found that polysaccharide (DvP-1) obtained from D. devonianum could activate macrophages in vitro, as evidenced by inducing morphologic change, thereby promoting the production of cytokines TNF-α, IL-6 and NO, and enhancing the pinocytic activity of macrophages ^[99]. Further study suggested that DvP-1 could activate macrophages through several toll-like receptors (TLRs), but mainly through TLR4. DvP-1 served as a TLR4 agonist and induced ERK, JNK, p38, and IκB-α phosphorylation, indicating the activation of MAPK and NFκB signaling pathways downstream of TLR4. Another polysaccharide (DDP) obtained from Dendrobium devonianum could also promote the immune functions of macrophages including NO release and phagocytosis ^[98]. These findings could help us further understand the immunomodulating effects of D. devonianum.

Investigations both in vivo and in vitro indicated that Dendrobium polysaccharides showed potential anti-cancer bioactivity. Polysaccharides DDP1-1, DDP2-1 and DDP3-1 (50, 25, 12.5 mg/kg body weight, respectively) isolated from D. denneanum were injected intraperitoneally into sarcoma 180 cell tumor-bearing Kunming mice for 14 days with cyclophosphamide (12.5 mg/kg body weight) as positive control. DDP1-1 showed higher effect than that of DDP2-1 and DDP3-1. Interestingly, the anti-tumor activity of DDP1-1 decreased from 72.04% to about 50.0%, with the increasing concentrations from 12.5 to 50 mg·kg^–1. In addition, DDP1-1 also could obviously promote the secretion of IL-2, TNF-α and IFN-γ and increase the immune function, which were considered to be the possible mechanism of anti-tumor effect ^[91]. Similarly, D. officinale polysaccharides (DP) could inhibit the S180 cell growth as well, while the possible mechanism was similar to that of DDP1-1. What’s more, it had cytotoxicity on hepatoma SMMC27721 cell line in vitro ^[116]. In another study, crude polysaccharide DDP from D. devonianum showed the inhibition effect on the S180 tumor-bearing mice possibly through enhancing the immune activity and increasing the colonic short chain fatty acids (SCFAs) ^[117]. DNP-W1 and DNP-W3 from D. nobile also showed the anti-tumor effects on S180 mice in vivo, besides, they could inhibit HepG2 and HL-60 cells growth in vitro ^[36].

After HepG2, A549, F9 and NCCIT tumor cells were treated with D. officinale polysaccharides (100–400 μg·mL^–1), the tumor cells growth were significantly inhibited, while the proliferation of splenocytes was promoted at the concentrations ranging from 25 to 200 μg·mL^{–1 [118]}. DCPP1a-1 was a homogeneous polysaccharide fraction from D. candidum. This polysaccharide also had anti-tumor effects on H22 tumor-bearing mice ^[71]. Compared with control group, DCPP1a-1 might impede xenografted tumor cells growth, while thymus and spleen index were higher at low dose (50 mg·kg^–1. Interesting, the antitumor effects at high dose were weaker than that of low dose treatment.

Crude polysaccharides of D. huoshanense (DHP) and its two homogeneous sub-fractions (DHP-1 and DHP-2) could significantly inhibit SGC-7901 cells growth in a dose- and time-dependent manner. Further study suggested that effect may be mediated by the decrease of the expression of gene c-myc and the increase of the expression of gene p53 ^[119]. Subsequently, DHPD1 was purified from crude polysaccharide of D. huoshanense and hydrolyzed with pectinase for 1, 8 and 24 h to yield three polysaccharides (designated DHPD1-1, DHPD1-8 and DHPD1-24, respectively) ^[120]. Study on the structure-activity relationship suggested that the DHPD1-24 was the core structure for DHPD1. Its lowest average molecular was approximately 1552 Da. This oligosaccharide might inhibit HLE cell apoptosis induced by H₂O₂ through the suppression of the MAPK signaling pathways ^[120]. Another interesting study indicated that combined use of D. candidum polysaccharide (DCP) with rIL-2 could dramatically enhance the killing activity of lymphokine activated killer cell (LAK) of peripharal blood (PB-LAK) and umbilical cord blood (CB-LAK) against cancer cells of patients in vitro ^[121]. Moreover, purified polysaccharides (DCPP-I, DCPP-I-a, DCPP-II) of D. chrysotoxum displayed the inhibition effects on proliferation of lung cancer cells (SPC-A-1 cell line) ^[122].

Extract of Dendrobium candidum (DCE) had anti-tumor property as well. Latest research indicated that it could suppress liver cancer SMMC-7721 and BEL-7404 cells as well as primary liver cancer cells likely though inducing mitochondria apoptosis and inhibiting Wnt/β-catenin pathway by blocking nuclear translocation of β-catenin ^[123]. Besides, DCE decreased colony formation, induced cell cycle arrest, and led to cell cycle-associated proteins ’ abnormal expressions in SMMC-7721 and BEL-7404 cells. DCE also effectively suppressed viability and proliferation of primary liver cancer cells and induced aberrant expressions of cell cycle- and apoptosis-related proteins. This finding could provide useful information for the clinical application.

Natural polysaccharides from Dendrobium showed good bioactivity with low or without toxicity. Therefore, the researchers aimed to study the effect of polysaccharides on injured liver induced by ethanol, carbon tetrachloride (CCl₄), selenium and so on to explore the correlation between the hepatoprotective effect and the potential mechanism. Histological examination was applied to evaluate the extent of liver damage. Some biochemical indexes were measured to evaluate liver function and hepatic lipid metabolism, such as MDA, glutathione (GSH), CAT, superoxide dismutase (SOD) and glutathione S-transferase (GST), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), 8-OHdG, total bilirubin (TBIL), total cholesterol (TC), low density lipoprotein (LDL) and high-density lipoprotein. In addition, the level of some cytokines and anti-inflammatory cytokines in serum initiating the inflammatory cascade of liver injury could be employed as indicators as well.

Male Kunming mice with CCl₄-induced liver injury were orally administered by homogeneous polysaccharide DHP1A from D. huoshanense for 14 days. This resulted in an obvious hepatoprotective effect through reversing the decrease in the expression of inflammatory cytokines (TNF-α and IL-1β), p-IκBα and CD68, and increasing in an anti-inflammatory cytokine (IL-10) with low hepatoxicity compared with the control group treated with CCl₄ ^[124]. Daily intragastrically administration of GGM, a homogeneous polysaccharide isolated from D. huoshanense, had a beneficial effect on selenium-induced liver injury and fibrosis ^[125]. On the one hand, GGM could decrease the activity of some biochemical indexes in serum and liver tissues including ALT, AST, LDH, and increase the amount of GSH with the increased dose. This polysaccharide could restore the activities of SOD, CAT and GST in a dose-dependent way (P < 0.05). On the other hand, histopathological changes initiated by excessive selenium intake were alleviated compared selenite control group. The potential mechanism involved might be that GGM could scavenge oxides induced by selenium and inhibit the expression of transforming growth factor-β1 (TGF-β1) and collagen in liver. Tian et al explored the hepatoprotective effects of four polysaccharides (DHP1, DHP2, DHP3 and DHP4) purified from young seedlings of D. huoshanense on CCl₄-induced acute liver injury. They found that DHP1 also significantly decreased the levels of ALT and AST in serum and the amount of hepatic MDA, and restored the activities of SOD and CAT after oral administration treatment ^[126].

Wang et al investigated hepatoprotective effects of the polysaccharide (DHP) from D. huoshanense on the basis of proteomics and metabolomics approaches on ethanol-induced liver injury in mice. The results showed that DHP could obvious ameliorate the altered metabolite levels of fatty acid, glycerophospholipid, amino acid and xenobiotics metabolism in both serum and liver tissue, and alleviate inflammatory and steatosis symptoms in liver histology ^[127]. In addition, the proteomic results indicated that orally administration of DHP could protect liver from injury induced by ethanol via adjusting the abnormal levels of carbohydrate, amino acid and lipid which could reduce cytotoxic methylglyoxal (MG). Especially, it could recover the disordered hepatic MG metabolism and methionine metabolism pathway perturbed by ethanol intake ^[128]. Besides, other scientists also observed the hepatoprotective effects of polysaccharides from various Dendrobium species with the similar mechanisms aforementioned ^[129].

There was one study focusing on investigating the protective roles and mechanisms of D. officinale polysaccharides (DOPS) on secondary liver injury in acute colitis ^[130]. The result indicated that DOPS could down-regulated TNF-α signaling pathway and activated Nrf-2 signaling pathway in vivo and in vitro. DOPS attenuated Dextran sodium sulfate (DSS)-induced hepatic pathological damage, liver parameters, infiltration of macrophages, cytokines levels, MDA level and increased the antioxidant enzymes activities. This demonstrated that DOPS might be a potential effective therapeutic reagent to attenuate secondary liver injury in acute colitis.

In recent years, plant polysaccharides such as inulin and fructooligosaccharides have gained great interest in nutrition and medical science for their prebiotic bioactivity. Previous study suggested that the polysaccharides from Dendrobium were difficult to be digested in the simulated gastrointestinal digestion ^[131]. Recent research investigated the effects of polysaccharides from D. officinale (DOP) ^[132] on human intestinal microbiota composition and metabolism via in vitro fermentation. Results showed that total carbohydrate from DOP could be digested by human intestinal microbiota during fermentation for 48 h. Meanwhile, the total short chain fatty acids (SCFAs) productions significantly increased. The major SCFAs were acetic, propionic and butyric acids, which were the results of the fermentation of polysaccharides and had many beneficial health outcomes for the host.

Dendrobium is one of the most important traditional Chinese medicinal foods used to treat age-related disorders. Research suggested that Dendrobium had the potential to provide neuroprotection against Alzheimer ’s disease related cognitive impairment via modulation of microglial activation ^[133]. Aged-related cognitive decline is a normal physiological process, which is an irreversible process that starts slowly but worsens over time, eventually leading to dementia. Alzheimer’s disease which has a pathology that is strongly linked with amyloid beta plaque (Aβ) and hyperphosphated Tau-formed neurofibrillary tangles in brain, is the most common type of dementia ^[134]. In vitro experiments showed that D. officinale polysaccharides (DOP) pretreatment contributed to BV2 cells shifting from proinflammatory to anti-inflammatory phenotypes with enhanced Aβ clearance in response to Aβ insults. For the in vivo study, the results showed that DOP remarkably attenuated cognitive decline in the senescence-accelerated mouse prone 8 (SAMP8) mice after the mice were chronically treated with DOP in drinking water from 4 to 7 months of age. DOP downregulated nterleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to inhibit the increased hippocampal microglial activation in SAMP8 mice, with the upregulation of interleukin-10 (IL-10), neprilysin (NEP) and insulin-degrading enzyme (IDE). In addition, the accumulation of hippocampal Aβ42 and phosphorylated Tau proteins in SAMP8 mice was also reduced.

Another study also found that that D. officinale polysaccharide (DOPS) had an appreciable therapeutic effect on learning and memory disabilities via up-regulating expressions of Nrf2/HO-1 pathway and inhibiting activation of astrocytes and microglia in ovariectomy- and D-galactose- induced cognitive decline ^[135]. This was the first time to demonstrate the effect and underlying mechanism of DOPS on the learning and memory impairment in both OVX- and D-gal-induced mice models.

Some natural and modified polysaccharides from Dendrobium species were found to be capable of enhancing anti-glycation activity. A homogeneous polysaccharide DHP- W2 from D. huoshanense could inhibit glycation in a dose- and time-dependent manner in vitro ^[55]. Otherwise, polysaccharides DHPD1 and DHPD2 were sulfated to yield the SDHPD1, SDHPD21 and SDHPD22 with the substitution degree (DS) of 1.473, 0.475 and 0.940, respectively ^[53-54]. Both the native and modified polysaccharides could reduce the formation of advanced glycation end products (AGEs) in the whole stage of glycation. The capability of inhibiting the formation of Amadori products was indicated to be in the order of SDHPD1 > DHPD1 > aminoguanidine, throughout the culture period when the agents were fixed at the same concentration. The peak inhibition of 80.2% was observed in the group with 1.0 mg·mL^–1 SDHPD1 treatment at the culture time for 21 day, which was 1.49-fold and 2.21-fold that of DHPD1 and aminoguanidine, respectively. As to DHPD2, after 28 days of incubation, SDHPD22 showed the highest inhibitory ability of 51.9% at the concentration of 1.0 mg·mL^–1, which increased by 18% and 47% than that of SDHPD21 and DHPD2 at the same dosage. The anti-glycation effect of sulfated polysaccharide was stronger than that of native polysaccharide, and polysaccharides with higher DS showed better activity than the lower ones. Structural analysis revealed that sulfation occurred at C-2 and C-6 of glycosyl residues in DHPD1 and DHPD2 was beneficial to enhance their anti-glycation activities. This suggested that the sulphuric group was important for the activity.

Sjögren’s syndrome (SS) usually described as a chronic autoimmune disorder of exocrine glands which results in xerostomia (dry mouth) and keratoconjunctivitissicca (dry eyes). It is therefore one-third of the most common autoimmune disorders ^[136]. The pathogenesis process of Sjögren’s syndrome (SS) includes the following main steps. In brief, glandular cells were active by external factors which would result to alteration of glandular vascular endothelium or dendritic cells ^[137]. Then the HLA-independent innate immune system would be activated followed by activation of lymphocytes with the gland leading to production of autoantibodies, cytokines, and metalloproteinases which probably causes dysfunction and apoptosis of the glandular tissue ^[138-139]. These factors might influence the abnormal distribution of aquaporin involving the water channel protein aquaporin-5 (AQP-5) in salivary and lacrimal gland ^[140-141]. The gland has an excess of receptors providing a target for the therapeutic ^[142]. The activation of both muscarinic receptors M1 and M3 can lead to optimum glandular secretion ^[143-144]. Hence, currently, there is no consensus treatment for SS. The treatment is generally aimed at ameliorating inflammation, inhibiting apoptosis of gland cells, alleviating the dryness symptoms and related morbidities and regulating abnormal expression of aquaporin ^[145]. Crude polysaccharide from D. officinale could reverse the pathological changes of SS in both cell and animal models ^[146-148]. The potential mechanism main implicated in the regulating the AQP-5 and maintaining its function might through suppressing the lymphocytes infiltration, pro-inflammatory cytokines secretion and subsequent apoptosis ^[149], stimulating the M3 muscarinic receptors leading to AQP-5 translocation to the apical membrane of human submandibular of gland epithelial cells which would promote saliva secretion ^[150]. However, more researches still need be carried out by using homogeneous polysaccharides, because the crude polysaccharide is difficult to be standardized.

It has been reported that polysaccharide from Dendrobium also exhibited positive effects on diabetes and diabetes-related diseases. It is well known that diabetes is a metabolic disorder with disturbance of protein, carbohydrate, and fat metabolism caused by the defects in insulin action, and insulin secretion, or both ^[151]. In one category, diabetes can be classified as type 1 diabetes leading to an absolute insulin secretion deficiency and type 2 diabetes with predominantly insulin resistance and an inadequate compensatory insulin secretory response ^[152]. Diabetes mice induced by alloxan were orally administrated with crude polysaccharides from D. huoshanense (DHP), D. nobile (DNP), D. officinale (DOP), D. chrysotoxum (DCP). They showed anti-diabetes activity with the order of DHP > DNP > DOP and protective effects against the damage in pancreas tissue except DCP ^[153]. Polysaccharide from D. moniliforme (L.) Sw. (DMP) has significant effects on adrenalin and alloxan induced diabetic mice which might be attributed to the decrease of serum glucose (P < 0.01) and the increase glucose tolerance but no significant influence on the serum glucose level of norm al mice ^[154]. The results were also found that crude polysaccharide from D. denneanum exhibited the similar effects on alloxan-induced hyperglycemia mice ^[89]. Polysaccharide from D. huoshanense could suppress the diabetic cataract via decreasing the expression of iNOS gene and advanced glycation end products (AGEs) ^[155]. The study by LI et al ^[156] also indicated that polysaccharide from D. officinale (PDC) exhibited the effect on diabetic retinopathy rats via reducing the expression of inflammatory cytokines [Interleuldn-6 (IL-6), TNF-α] and restraining the vascular endothelial growth factor (VEGF) expression which might be associated with NF-κB signal pathway. Another study also found that polysaccharide from D. officinale had remarkable hypoglycemic effect in streptozotocin (STZ)-induced type 2 diabetic BALB/c mice ^[157]. The hypoglycemic effect of DOP might be associated with the regulation of glycogen synthesis and the activity of glucose metabolism enzymes via activating the PI3K/Akt signaling pathway. Latest study by Wang et al showed that the purified GXG from D. huoshanense polysaccharides had the capability of ameliorating hyperglycemia and improving insulin sensitivity in T2D mice ^[158].

Dendrobium is a precious herb medicine and widely used in China and Southeast Asian countries to “nourish stomach”and enhance production of body fluid for thousands of years. One study was the first report to demonstrate the crude polysaccharide obtained from D. officinale which had the gastroprotective effect through inhibiting oxidative stress-induced apoptosis ^[159]. In addition, authors found that polysaccharide from Dendrobium nobile (JCP)could protect against the ethanol-induced gastric ulcer ^[160], and the biological activities of polysaccharides depend on their structure characters.

Recent research suggested that D. officinale polysaccharide (DOP) could ameliorate the inflammation and enhance lung functions while remarkably down-regulate mucin-5AC and up-regulate AQP-5 expression in both chronic obstructive pulmonary disease (COPD) models and patients by use of Western blot and qPCR techniques ^[161]. Also D. officinale polysaccharide (DOP) was found to relieve ovarian damage through the protection of mitochondria in the ovaries ^[162].

Furthermore, the polysaccharide from cultivated Dendrobium huoshanense (cDHP) could resist cigarette smoke (CS)-induced lung inflammation ^[59]. In addition, polysaccharides (PDO) obtained from D. officinale could significantly ameliorated indices for both pulmonary inflammation and fibrosis in a bleomycin (BLM)-induced pulmonary fibrosis model in rats, which was associated with inactivation of transforming growth factor β1 (TGFβ1)-Smad2/3 signaling pathway. In addition, PDO effectively blocked TGFβ1-induced transformation of rat alveolar epithelial type II cells into myofibroblasts, with the inhibition of total Smad2/3, pSmad2/3, collagen I and fibronectin protein expression in a dose-dependent manner in vitro ^[163]. Therefore, PDO may function as a promising candidate compound for therapy of idiopathic pulmonary fibrosis.

What’s more, recent studies had shown that Dendrobium officinale polysaccharide (DOP-GY) had the potential to exert cardioprotective effects against H₂O₂-induced H9c2 cardiomyocyte apoptosis ^[164]. Not only that, polysaccharide with acetyl group from D. officinale was beneficial to the colonic health ^[165]. To sum up, polysaccharides from Dendrobium have many biological activities and deserve our attention and more exploration.

Like proteins and nucleic acids, polysaccharides also play an important role in organisms. For instance, heparin, lentinan and ganoderan have been using in a wide range in clinical. Polysaccharides widely exist in plants and animals, which provides a wide range of material base for the research. Recent decades, Dendrobium polysaccharide as a very valuable one attracting more attention of the researchers for it has very high medicinal value. Polysaccharides from Dendrobium species have been found mainly to be composed mannose and glucose, while a small amount of galactose also exist. (1→4)-linked glycosyl residues frequently occurred in the homogeneous polysaccharides which usually were substituted by acetyl groups with different degree at O-2 or/and O-3 position of mannose and O-2 or O-3 of glucose as well. Investigation about biological activity suggested that polysaccharides from Dendrobium showed multiple bioactivities with low or without toxicity, including anti-oxidant, anti-tumor, anti-diabetes, anti-glycation, immunomodulatory function, hepatoprotective effect, promoting the secretion of glands, etc.

Preliminary structure-activity relationship studies indicated that the existence of acetyl groups was beneficial to bioactivities, while influence of the exact substitution degree and sites on the activity is not known yet. This could be a direction of future research. Currently, there are still some challenges in the research of Dendrobium polysaccharides. Firstly, what’s the core structure of polysaccharides underlying biologically activities? This will provide the basis for chemical synthesis of the active polysaccharides. This also provides convenience for clinical research as well as important references for the study of the active polysaccharides. In addition, polysaccharides isolated from stems are usually polluted by starch, which make it hard to be purified. Similarly, polysaccharides from Dendrobium species usually contains 1, 4-linked glucosidic bond which might be hydrolyzed during the process of enzymolysis of amylase. However, the chemical shift of (1→4)-linked mannose and glucose very close to each other, which make the structure elucidation more difficult.

Secondly, the existence of acetyl groups and the relatively high viscosity of some polysaccharides solution make it difficult to be characterized. Present structure analysis main focused on monosaccharide composition, sugar linkage patterns, main chain and the side chain, however, the more- advanced structure still under elucidating. Besides, the functional target molecules of polysaccharide from Dendrobium species are largely unknown.

Thirdly, deeply researches of structure-activity relationship of polysaccharides still need to be carried out for many researches were based on crude polysaccharide. Hence, researches about bioactivities and their mechanism of homogenous polysaccharides are more valuable. However, more experiments in vivo also need to be investigated.