Sacchachitin, a novel chitin-polysaccharide conjugate macromolecule present in Ganoderma lucidum: Purification, composition, and properties

Abstract

Context: The extraction method and the crude wound healing effects of sacchachitin from Ganoderma tsugae Murr. (Ganodermataceae) has been cited. However, its purity is still largely limited.

Objective: An improvement of the fractionation protocol to purify the sacchachitin from Ganoderma lucidum L. (Ganodermataceae) (SGL) is needed.

Methods: Fruiting bodies were extracted with double distilled water and subsequently the residue treated with 95% ethanol and then 40% ethanol. After being filtered, the pH of the supernatant was adjusted to 4.0 with 1 N HCl and lyophilized. The supernatant was added (3:1 v/v) ethanol, the precipitate was collected, 2% NaOH was added and refluxed. The supernatant was collected with pH adjusted to 4.0, then treated with 10% potassium hydroxide (KOH) with repeating acid precipitation and (3:1) ethanol precipitation twice more to obtain the sacchachitin.

Results: SGL had a hexosamine content 16.3% (w/w), firmly linked to a talomannan. Its Fourier Transform Infrared Spectroscopy (FTIR) spectrum revealed specific absorption (in cm^–1) ν_O–H 3455.5 b,s, amide ν_C=O 1678.5, and amide I° δ_N–H 1550.4. The percentage deacetylation degree was 37.6 and 39.4% for SGL and MSC, respectively. As contrast, MSC contained only 6.6% of hexosamine with a low protein/carbohydrate ratio 0.35 comparing to 0.82 for SGL. SGL was only moderately strong antioxidant regarding the anti-DPPH, antihydroxyl free radical, and antisuperoxide anion capabilities, exhibiting an IC₃₃ values of 10 mg/mL (the highest scavenging capability never exceeding 33%), 0.9 mg/mL, and 4.8 mg/mL, respectively.

Conclusion: We have successfully isolated the pure sacchachitin from the fruiting bodies of G. lucidum that exhibits potent antioxidative activity and may be useful in fabrication of the artificial skin composite substitute.

Keywords::

• IR spectrum
• wound healing
• biomedical materials
• talomannan
• antioxidant
• artificial skin

Introduction

Ganoderma lucidum L. (Ganodermataceae) enjoys special veneration in Asia, where it has been used as a medicinal mushroom in Traditional Chinese Medicine for more than 2000 years, making it one of the oldest mushrooms known to have been used in medicine (Sanodiya et al., 2009). It belongs to basidiomycete white rot fungus mostly used as an official medicine in many provinces of China (Lee, 1578) and other Asian countries including Japan and Korea (Russell & Paterso, 2006). G. lucidum is famous for its folkloric names Língzhī (in Chinese), or Munnertake, Sachitak, or Reishi (in Japanese), or Yeongji (in Korean). In Chinese, the word Língzhī actually means “the herb of spiritual potency” or “the mushroom of immortality” (David, 1986). But as often noted, Língzhī commonly denotes one general form for mushroom G. lucidum and its close relative Ganoderma tsugae Murr.

Preclinical studies have established that the polysaccharide fraction of G. lucidum has a diversity of potent medicinal effects (Russell & Paterso, 2006). Currently, Língzhī has been officially listed in the American Herbal Pharmacopoeia and Therapeutic Compendium.

The term “sacchachitin” was first used by Su et al. (1997) for description of special glucosamine derivative isloated from G. tsuga. This Su’s sacchachitin is a copolymer made from β-1,3-glucan (approximately 60%) and N-acetylglucosamine (approximately 40%), having a filamental structure. This pulpy white residue was tested for use as a skin substitute, thereby wound healing was found faster, compared to the commercialized skin substitute, the chitin sheet made of crab shell (Beschitin^TM). Furthermore, many researchers indicated that sacchachitin exhibits potent antibacterial (Yang et al., 2001; Lin, 2001; Wang & Ng, 2006; MoradaLi et al., 2006) and antiviral activities (Wang & Ng, 2006; MoradaLi et al., 2006).

Accumulating studies have shown that Ganoderma mushrooms possess antitumor, immunomodulatory, immunotherapeutic, antiagglutination (Liu et al. 2006), hypotensive, hypocholesterolemic, and hypoglycemic bioactivities (Bensky et al., 2004). Ganoderma mushrooms are useful preventive nutraceutics against cancer metastasis (Lee et al., 1984), its potency is comparable to Lentinan prepared from Shiitake mushrooms (Suga et al., 1984). The mechanism by which G. lucidum affected cancer is still unclear. Different cancer stages can be affected differently. In general, it affects the cancer development by inhibiting angiogensis, migration, metastasis of the cancer cells, and inducing apoptosis of tumor cells (Paterson, 2006). Moreover, the Ganderma compounds inhibit 5α-keto-reductase activity that is responsible for the biosynthesis of dihydrotestosterone (Liu et al., 2006), implicating its therapeutic value in prostate cancer.

Its active constituents include polysaccharides, terpenes, and others isolated from the fruiting bodies and mycelia of this fungus (Paterson, 2006; Lindequist et al., 2005). Ganoderic acid is a moderate hepatoprotective agent (Li & Wang, 2006). In addition, Ganderma-produced sterols inhibit lanosterol 14α-demethylase activity required for the biosynthesis of cholesterol (Hajjaj et al., 1986).

Zhu et al. (2007) found G. lucidum polysaccharides enhanced the function of immunological effector cells in immunosuppressed mice. More recently, Chen et al. (2009) indicated that the polysaccharides obtained from the fruiting bodies of G. lucidum effectively scavenged free radicals of DPPH, superoxide anions, and hydroxyls in vitro, and activated the antioxidative enzymes like SOD, GPX, and CAT, and enhanced immunity as indicated by levels of IL-1β, IL6, and TNF-α in rats affiliated with uterine cancer.

Materials and methods

Chemicals

Deinoinzed water, ethanol (95%), ethanol 40%, NaOH (2%), KOH (10%), H₂O₂ (5.6%), anhydrous sodium carbonate, acetylacetone, ethanol, authentic glucosamine HCl, p-dimethylaminobezaldehyde, absolute ethanol, and 95% ethanol were purchased from Wako Pure Chemicals (Osaka, Japan). For determination of hexosamine, the following reagents and solutions were prepared: 1.5 N Na₂CO₃ solution and 4% acetylacetone solution in 1.5 N Na₂CO₃. These two solutions must be freshly prepared before use. Ehrlich reagent: 0.267 g of p-dimethylaminobenzaldehyde were dissolved in 10 mL of mixed solvent of 95% ethanol: HCl (=1:1) to make 2.76% Ehrilch reagent. Standard glucosamine HCl solution: 0.2407 g of glucosamine HCl were accurately weighed and dissolved in 10 mL water to obtain a final concentration of 20000 µg/mL (stock solution 1), 50 mL of which was diluted with double distilled water to make a total volume of 1000 mL (stock solution 2, c = 100 μg/mL). These stock solutions were stored in a 4°C refrigerator immediately when prepared.

Source of the fruiting bodies of G. lucidum

The fruiting bodies of G. lucidum were purchased from the local Ganoderma supplier in Hsin-Chu County of Taiwan. The classification of taxonomy was carried out by Dr. C. H. Su with the Fungal Strain Research and Protection Lab, Food Industrial Research Centre, in which the voucher has been filed out.

Proximate composition analysis

The proximate analysis was performed according to the methods described in the Association of Official Analytical Chemists (AOAC, 1995).

Fractionation of polysaccharides and preparation of sacchachitin

The process shown in Figure 1 was followed to isolate and purify the sacchachitin from the fruiting bodies of G. lucidum L (SGL). In parallel, Su et al. (1997) was followed to prepare the contrast sample “mimic sacchachitin Su et al. (MSC)”.

Figure 1.  Flowchart showing the process used for purification of sacchachitin from G. lucidum.

FTIR spectra of the SGL and MSC

The KBr (IR grade) tablet of sample was prepared. Briefly, the two sacchachitin samples (each 50 mg) were separately mixed with KBr powder (IR grade) at a ratio KBr/sacchachitin = 200:1, macerated thoroughly to obtain a completely homogeneous state and then the KBr/sacchachitin tablet was fabricated. The IR spectrum was taken with the Nicolet Type Protege 460 IR dual beam Spectrophotometer ESP against the KBr blank. The running conditions were: accumulation, 5; resolution, 4 cm^–1 zero filling, ON; apodiation, cosine; gain, Auto (David, 1986); scanning speed, Auto (2 mm/s).

Degree of deacetylation

The degree of deacetylation (DD) of these two isolated products was determined according to Lin et al. (1992). Briefly, desiccated chitin samples (each 0.5 g) were accurately weighed and thoroughly dissolved in 0.1 M HCl. The solution was then titrated with 0.1 M NaOH. The degree of deacetylation was calculated according to Eqs 1 and 2:

1

where C₁ is the concentration of HCl (in M). C₂ is the concentration of NaOH (in M). V₁ is the volume of 0.1 M HCl (in mL). V₂ is the volume of 0.1 M NaOH consumed (in mL); G, the weight of chitin sample. W is the water concentration of chitosan sample (in %), and 0.016 is the weight of NH₂ equivalent to 1 mL 0.1 M HCl (aq). Substitution of the percentage of functional NH₂ obtained from Eq. 1 into Eq. 2 to obtain

2

where 9.94 is the theoretical NH₂ percentage equivalent.

Assay for carbohydrate

The content of carbohydrate was determined by the phenol-H₂SO₄ method.

Assay for protein

Protein (% w/w) in each extract was determined by the Bradford protein assay (Ker et al., 2010).

Assay for hexosamine

Hexosamine content (% w/w) in each extract was determined by the Johnson (1971) hexosamine assay.

The monosaccharide composition in partially purified sacchachitin (PPS)

Hydrolysis, reduction, and derivatization of monosaccharides

The method of Ker et al. (2010) was used; in principle, the PPS was subjected to complete acid hydrolysis. The hydrolyzed mixture of monosaccharides was acetylated to form acetylated monosaccharides and analyzed with GC/MS. The quantity in mol % of each individual monosaccharide was calculated from the corresponding percent peak area by comparing with the blank. Practically, the method of Ker et al. (2005) was followed. PPS (2 mg) was accurately weighed and transferred into the Cole–Parmer reactor, to which 2 mL of 2 M trifluoroacetic acid was added. The mixture was heated at 120°C with the Cole–Parmer heater for 24 h while shaken vigorously every 30 min during heating until completely hydrolyzed. The hydrolyzed mixture was subjected to a stream of nitrogen until dried. The residue was dissolved twice with NH₄OH solution. Each time 100 μL of 1 M NH₄OH containing 1 mg/mL of deoxyribose was used as the internal standard. The reduction was conducted in the Cole–Parmer reactor with 0.2 g of NaBH₄/10 mL of DMSO. The remaining procedures were conducted following Ker et al. (2005). The final deduced product was transferred into a 1 mL reaction vessel, lyophilized and analyzed with GC/MS as described (Ker et al., 2005).

GC/MS analysis

GC/MS was conducted in the Restek Rtx 225 column (l × i.d. = 30 m × 0.32 mm). The temperature was programmed, initially at 60°C for 1 min, raised at an elevation rate 8°C/min until 220°C, and held at this temperature for 10 min. The temperatures at the injection port and the detector were set equally at 230°C. Hydrogen was used as the carrier gas and operated at a flow rate of 2.0 mL/min. The reference values of retention time of the standard monosaccharides in Ker et al. (2010) are followed.

Preparation of sample solution

Glucosamine HCl powder (10 mg) was accurately weighed and transferred into a 250 mL Erlenmeyer flask. To the sample, 80 mL of hot double distilled water (80°C) was added and stirred vigorously while heating on the heater at 90–100°C until dissolved. The solutions were made to 100 mL with double distilled water to obtain the sample solution (c = 100 µg/mL).

Preparation of standard glucosamine solution

The stock solution 2 of authentic glucosamine (c = 100 μg/mL) was successively diluted to obtain 5, 10, 25, 50, 100, and 250 μg/mL, respectively.

Establishment of calibration curve

The glucosamine HCl standard and sample solutions (0.5 mL each) were respectively transferred into test tubes having screw type capping. To each tube, 0.5 mL of freshly prepared acetylacetone solution was added. After capping, the reaction mixture was incubated at 100°C in a water bath for 30 min when the reaction was completed; the reaction fluids were cooled to room temperature for 10 min. To each tube, 2.5 mL of absolute alcohol was added and mixed well, and then added 0.5 mL of freshly prepared Ehrlich reagent. The mixtures were agitated and incubated at 60°C for 25 min to facilitate the color formation (pink color). On standing for 10 min at ambient temperature, the optical density was read at 528 nm.

Mean molecular weight of different fractions

The method originally reported by Yagi et al. (1986) [later slightly modified by Ker et al. (2010)] was used for determination of the mean molecular weight (MW) of polysaccharides by gel permeation chromatography (GPC). Briefly, to 20 mg of each fraction (L1–L10) and the “mimic sacchachitin Su et al.”) 1 mL of 10% NaOH and sufficient double distilled water were added to make a final volume of 5 mL. The mixture was thoroughly agitated to facilitate the dissolution and centrifuged at 2500 rpm for 10 min to remove the insoluble precipitate. The supernatant was decanted, each 3 mL of which was eluted with 0.05 N NaOH solution containing 0.02% NaN₃ at a flow rate 0.5 mL/min on a Sephadex G-100 column (id × ℓ = 2.5 × 100 cm). The fraction collector (ISCO Retriever 500, Isco., Lincoln, NE, USA) was used to collect the eluents, 6 mL per tube. The collection was stopped at the 100th tube. The calibration curve was established using authentic dextrans having molecular masses of 8.8, 40, 500, 2000, and 5000–40 000 kDa (Sigma, St. Louis, MO, USA), respectively. The molecular mass distribution and the average molecular mass was calculated against this linear correlation between the logarithm of each standard molecular mass and the ratio the eluted to the void-volume (Ker et al., 2010).

Calculation

The linear correlation of standard glucosamine solution has two linear ranges which is feasible for the concentration range 0–100 µg/mL (Eq. 3) and 0–250 μg/mL (Eq. 4), respectively.

3

(R² = 0.9975, feasible for concentration range 0–100 μg/mL).

4

(R² = 0.9996, feasible for concentration range 0–250 μg/mL).

Results and discussion

Proximate composition of the whole fruiting bodies

The proximate composition of the G. lucidum fruiting bodies was very similar to many other common mushrooms, particularly regarding the low fat (3.0%), high crude fiber (29.2%), and carbohydrate (41.6%) and moderate ash content (1.5%) (Table 1), implicating the presence of huge amount of valuable nutrients and mineral ions (Ouyang et al., 1998; Ma & Zhang, 2004).

Table 1.  Proximate composition in the fruiting bodies of G. lucidum.

Composition	Content (%w/w)
Moisture	11.0 ± 0.3
Crude ash	1.5 ± 0.1
Crude fat	3.0 ± 0.2
Crude fiber	29.2 ± 0.3
Crude protein	13.6 ± 0.1
Carbohydrate	41.6 ± 0.7

Values are expressed as mean ± SD from triplicate experiments (n = 3).

A review by Lindequist et al. (2005) indicated that many edible mushrooms exhibit a broad spectrum of bioactivities that are beneficially acting as a complementary alternative medicine (CAM). As often cited, the therapeutic efficacy of mushrooms includes antibacterial, antifungal, antimultiresistant bacterial, antiviral, immunomodulating, antitumor or antitumor adjuvant, cytostatic, immunosuppressive and antiallergic, antioxidative and anti-inflammatory, antiatherosclerogenic, hypoglycemic, hepatoprotective, and neurotrophic functions (Lindequist et al., 2005).

Characterization of each fraction

Following the purification process (Figure 1), we obtained subsequently fractions L5–L10 (SGL) (Figure 1). The highest and the lowest yields were 9.1 and 0.7% for fractions L6 and L5, respectively. L10 was only moderately yielded (3.4%) comparing to 14.7% of MSC (Table 2). Uniquely, L10 (SGL) contained a very high level of hexosamine (16.3%). The hexosamine content decreased in the order L9 (11.1%) >L8 (8.9%) >L7 (8.6%) >L5 (6.3%) >L6 (4.6%). Well known to all, the hexosamine content always is acting as a purity index of chitins (Cardenas & Miranda, 2004). As reference, in fungal biomass the chitin yield was ranging within 8.5–19.6% (% dry basis), exhibiting a degree of acetylation (AD) 91.0–98.7%; a level of glucosamine and ash content (% of chitin) 2.4–8.2 and 26.4–64.5%, respectively (Di Mario et al., 2008).

Table 2.  Comparison of composition in different fractions and the sacchachitins obtained by different processes from the fruiting bodies of G. lucidum.

Fraction	Yield (%w/w)	Carbohydrate (%w/w)	Protein (%w/w)	Hexosamine (%w/w)	Mean MW, (kDa)
L5	0.7 ± 0.1^a	75.1 ± 0.3^f	21.4 ± 0.1^e	6.3 ± 0.1^b	8230 ± 58^e
L6	9.1 ± 0.2^e	52.9 ± 0.2^d	46.9 ± 0.2^g	4.6 ± 0.1^a	1101 ± 55^a
L7	1.5 ± 0.1^b	70.3 ± 0.3^e	28.1 ± 0.1^f	8.6 ± 0.1^d	1198 ± 56^b
L8	7.4 ± 0.2^d	41.8 ± 0.2^c	13.4 ± 0.1^c	8.9 ± 0.1^e	6945 ± 61^d
L9	3.5 ± 0.1^c	80.7 ± 0.3^g	15.6 ± 0.1^d	11.1 ± 0.2^f	4570 ± 44^c
L10: Sacchachitin (this paper)	3.4 ± 0.1^c	32.4 ± 0.2^a	10.1 ± 0.2^b	16.3 ± 0.1^g	8632 ± 62^f
Sacchachitin Mimic Su et al. (1997)	14.7 ± 0.3^f	28.0 ± 0.3^b	9.6 ± 0.1^a	6.6 ± 0.2^c	9756 ± 55^g

L5: ethanol precipitate from hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10 (SGL): Sacchachitin carbohydrate was measured by the phenol-H₂SO₄ method. Protein was determined by the Bradford protein assay. Hexosamine was determined by the Johnson hexosamine assay (Johnson, 1971). The molecular weight (MW) was determined by gel permeation chromatography (GPC). Values in each column with different superscripts indicate significantly different between each other (p < 0.05). Data are expressed in mean ± SD from triplicate experiments.

Further, when simultaneously taking the protein/carbohydrate ratio (P/C ratio) into consideration, the fractions L5, L7, L8, and L9, that respectively have P/C values 0.29, 0.40, 0.32, and 0.19, were classified as the category of peptidoglycan (Ker et al., 2010), while fractions L6 and L10 (SGL) having P/C values 0.89 and 0.82 respectively were identified to be glycoproteins. Based on this criticism, the MSC having a hexaosamine content 6.6% (w/w) and a P/C ratio 0.35 was confirmed to be merely one kind of peptidoglycan. In fact it was not a real sacchachitin (Table 2). Obviously, all data obtained are pointing to the chitin characteristics of fraction SGL.

The MW of all fractions ranged within 1101–8632 kDa, in which fractions L6 and L7 had the lowest MW, SGL had the medium MW 8632 kDa, comparing to the highest 9756 kDa of MSC (Table 2).

In reality, the MW of fungal polysaccharides depends on the cultivation condition. In liquid fermentation with the rice spirit waste water plus glucose, Hsieh et al. (2005) obtained polysaccharides form G. lucidum that had MW only ranging within 10–200 kDa, being far smaller than what we have found (Table 2). As can be expected, in solid cultivation the counter gravity force required would be far greater than those needed in the liquid cultivation. Moreover, although the molecular distribution pattern in both the intracellular and extracellular portions was very similar, the latter always exhibit higher MW (Hsieh et al., 2005), an implication in the main role of polysaccahcride acting as the basic supporting substratum for fungal fruiting bodies.

IR spectra of the sacchachitins and their relevant sacchachitosans

The characteristic absorption in Figure 2 reads (in cm^–1): ν_O–H at 3455.7–3450.5, ν_N–H 3455.7–3450.5, ν_C–H 2932.5–2932.4, 2300–2400 unassigned, ν_C=O 1678.5–1678.3, δ_N–H amide I 1551.4–1550.4, δ_C–H 1413.5–1424.5, ν_C–C 1154.6–1152.7, ν_C–N 1125.9–1121.7, and ν_C–O 1029.5–1028.7, mostly similar to the functionalities of FTIR spectra cited for chitosan (Cardenas & Miranda, 2004). The strong absorption band at amide ν_C=O 1678.5–1678.3 cm^–1 evidences the highly acetylated nature of L10 sacchachitin (Figure 2A; Table 3). Similar IR spectra were found for the completely deacetylated products (Figure 2B and 2C; Table 3) except that the absorption at amide ν_C=O 1678.5–1678.3 cm^–1 and amide I δ_N–H 1551.4–1550.4 cm^–1 have completely disappeared (Figure 2B and 2C; Table 3).

Table 3.  Assignment of IR absorption for different products and their deacetylation degree.

Product	Bond vibration frequency (cm^–1) found in the IR spectra
Vibration mode	Sacchachitin		Sacchachitosan
	This paper	MSC	This paper	MSC
νO–H	3450.5 b, s	3455.7 b, s	3445.2 b, s	3419.2 b, s
νN–H	3500–3300 (masked)	3500–3300 (masked)	3500–3300 (masked)	3500–3300 (masked)
νC–H	2932.5 w	2932.4 w	2920.7 w	2922.6 w
νC=O	Amide 1678.5	Amide 1678.3	Disappeared	Disappeared
δN–H	Amide I 1550.4	Amide I 1551.4	Disappeared	Disappeared
δC–H	1413.5;	1412.6;	1420.3;	1421.3;
	1381.6;	1376.4;	1384.6;	1373.1;
	1325.8	1324.5		1314.3
νC–C	1152.7	1154.6	1154.2	1158.4
νC–N	1125.9	1121.7	1123.5	1125.6
νC–O	1029.5	1028.7	1081.9	1078.0
δN–H	780.2	779.5	–	–
δC–H	712.3	713.3	–	–
δC=O	690.2	690.5	–	–
Deacetylation degree (%)^a	37.6 ± 2.6	39.4 ± 3.2	ue	ue

Source of the products: MSC was the sacchachitin processed according Su et al. (1997) from G. tsugae.

^aThe method for determination of the degree of deacetylation was following Lin et al. (1992).

b, s: broad and strong; w: weak. ue: unexamined.

Figure 2.  The IR spectra of sacchachitins and sacchachitosans. (A) Our product sacchachitn and MSC (the mimic Su et al., 1997). (B) Our product sacchachitosan and (C) sacchachitosan from MSC. The characteristic absorption reads in (A) (at cm^–1): ν_O–H 3455.7–3450.5, ν_N–H 3500–3300, ν_C–H 2932.5–2932.4, 2300–2400 unassigned, ν_C=O 1736.3–1734.6, amide I δ_N–H 1551.4–1550.4, δ_C–H 1413.5–1324.5, ν_C–C 1154.6–1152.7, ν_C–N 1125.9–1121.7, ν_C–O 1029.5–1028.7, δ_N–H 780.2–779.5, δ_C–H 713.3–712.3, and δ_C=O 690.5-690.2. Similar absorption was seen in (B) and (C), but lacking the bands ν_C=O 1736.3–1734.6 and the amide I bands δ_N–H 1551.4–1550.4.

Degree of deacetylation

The transformation of chitin to chitosan can be accomplished by either the deacetylation through the action of deacetylase (Cardenas & Miranda, 2004) or exhaustive alkaline hydrolysis (Champagne, 2008).

The DD of the L10 sacchachitin and MSC was found to be 37.6 and 39.4%, respectively (Table 3).

In chitins, N-acetylation can facilitate the intramolecular hydrogen bonding to be formed between the intramolecular C₃-OH hydroxyl to the C₅-O ring oxygen across each β(1→4)-glycosidic linkage, restricting the chitin units to remain at the low-energy chair conformation and creating a rigid and linear polymer backbone (Champagne, 2008). Consequently, the N-acetylation would reduce the solubility of chitin and affect the hydrogen bonding formation (Figure 5).

Pharmaceutically, poor solubility raises much limitation in clinical application of drugs. To increase the solubility in aqueous and organic solvents, chemical modification of chitin to generate new biofunctional materials is of primary interest. A very elaborate procedure includes alkaline N-deacetylation of the N-acetamido functional groups of chitin to produce chitosan. Modification of the N-acetamido groups by N-deacetylation results in functional amines that can undergo nucleophilic substitution reactions (Champagne, 2008). We speculate that in a highly acetylated state, many –OH groups are orderly lined up in a state firmly bound by hydrogen bonding in the low-energy chair conformation exhibiting a rigid and linear polymer backbone (Champagne, 2008). In highly deacetylated state, these –OH bonds instead are randomly exposed, and these released –OH groups no longer contact closely, resulting in reduced ν_O–H absorption at 3455.7–3450.5 cm^–1 from %T 27 to %T 37 (Figure 2A and 2B; Table 4). In contrast, the MSC that originally had a %T 36.5 at ν_O–H 3455.7–3450.5 cm^–1 retained at %T 3.5 after complete deacetylation. Such a phenomenon was also found for the δ_N–H bending absorption at 1640–1550 cm^–1. The %T of sacchachitin (this paper) was increased, but that of MSC decreased (Figure 2A–2C; Table 4). All of these data indicate the nonchitin character of MSC.

Table 4.  %T of characteristic IR absorption bands before and after deacetylation.

Vibrating bond	Frequency^a (cm^–1)	Sacchachitin		Sacchachitosan^c
		(This paper)	MSC^b	(This paper)	(MSC)
νO–H	3650–3300	3450.5	3455.7	3445.2	3419.2
%T	–	27.0	36.5	37.0	3.5
νC=O	Amide 1680–1630	1678.5	1678.3	Disappeared	Disappeared
%T	–	37.0	44.0
δN–H	1640–1550	1645.9	1637.3	1645.9	1637.3
%T	–	40.5	47.0	52.5	17.3
δN–H	Amide I 1570–1515	1550.4	1551.4	Disappeared	Disappeared
%T	–	41.0	47.5	–	–

^aCited frequency range.

^bMSC: the mimic sacchachitin (Su et al., 1997).

Saccachitosans were prepared from corresponding sacchachitins according to Lin et al. (1992) in this paper.

Immunologically, higher DD exhibits stronger break strength and activates higher collagenase activity, and histologically, more prominent proliferation of fibroblasts (Minagawa et al., 2007). Minagawa et al. (2007) hypothesized the indispensable role of free amino groups in the chitin/chitosan molecules. It has been reported that chitin with a 70% DD is most effective for macrophage activation (Nishimura et al., 1986). Obviously, in view of practical application, these two products were in underdeacetylated forms (Table 3), and further mild enzymatic deacetylation treatment is required for transforming the low DD to high DD state.

Monosaccharide pattern of different fractions

A total of eleven monosaccharides including rhamnose, fucose, ribose, arabinose, xylose, allose, talose, mannose, galactose, glucose, and myoinositol was found in the fruiting bodies of G. lucidum (Table 5). Amazingly, ribose was almost undetected in fractions L5–L9. Allose was undetected in fractions L6–L8, and myoinositol was only absent in fraction L7. Alternatively, extremely high content of talose and mannose were present in these two sacchachitins (Table 5). The amount reached 29.41 and 33.82 mole% for talose; and 43.92 and 47.90 mole% for mannose in the L10 (SGL) and MSC, respectively (Table 5). The glucose content was high in fractions L5–L9, particularly in fraction L8, which reached 73.48 mole%. However, glucose was extremely low in these two sachachitin peparations, giving only 1.87 and 0.69 mole% respectively in L10 (SGL) and MSC (Table 5). Characteristically, these two sacchachitins contained very high levels of talose and mannose, indicating the sacchachitins are firmly linked to talomannans in G. lucidum. Conversely, these two sacchachitins consisted of relatively low concentrations of galactose and myoinositol (Table 5). Taken together, the fact pointing to the unusually low content of hexosamine in MSC (Table 2), and the unusual IR spectral change before and after deacetylated (Table 4), we suspect that the magic anti-inflammatory and wound healing effect of MSC originally reported by Su et al. (1997) must have been contaminated by a huge amount of peptidoglycans coexisting in G. tsugae (Table 2).

Table 5.  Pattern of monosaccharide composition in different fractions obtained from the fruiting bodies of G. lucidum.

Fraction	Mole% in each fraction or in different sacchachitins
Sugar	L5	L6	L7	L8	L9	Sacchachitin (this paper)	MSC
Rhamnose	11.36 ± 0.07^e	11.15 ± 0.07^e	25.71 ± 0.12 ^a	6.41 ± 0.05^b	8.51 ± 0.07^c	4.49 ± 0.02^a	9.42 ± 0.07^d
Fucose	3.58 ± 0.02^f	3.15 ± 0.02^e	3.46 ± 0.02^f	0.14 ± 0.01^a	1.41 ± 0.01^d	0.59 ± 0.01^b	1.06 ± 0.02^c
Ribose	Trace	Trace	Trace	Trace	Trace	1.68 ± 0.02^b	0.86 ± 0.02^a
Arabinose	4.83 ± 0.02^d	5.83 ± 0.02^e	5.57 ± 0.02^e	0.35 ± 0.01^a	3.52 ± 0.02^c	6.12 ± 0.04^f	0.59 ± 0.02^b
Xylose	0.48 ± 0.02^b	3.08 ± 0.05^d	6.15 ± 0.10^f	0.31 ± 0.01^a	3.31 ± 0.06^e	3.31 ± 0.02^e	0.78 ± 0.01^c
Allose	1.58 ± 0.01^b	ud	ud	ud	0.79 ± 0.02^a	7.75 ± 0.11^d	3.32 ± 0.03^c
Talose	33.28 ± 0.12^e	32.68 ± 0.07^d	6.92 ± 0.10^a	6.83 ± 0.10^a	19.78 ± 0.10^b	29.41 ± 0.14^c	33.82 ± 0.13^e
Mannose	1.93 ± 0.02^c	0.88 ± 0.04^b	2.35 ± 0.01^d	0.86 ± 0.04^b	0.47 ± 0.01^a	43.92 ± 0.22^e	47.90 ± 0.27^f
Galactose	2.66 ± 0.04^e	2.64 ± 0.04^e	2.91 ± 0.03^f	0.69 ± 0.01^a	1.45 ± 0.02^d	0.71 ± 0.01^b	0.77 ± 0.01^c
Glucose	31.50 ± 0.23^c	35.38 ± 0.13^d	46.44 ± 0.28^e	73.48 ± 0.14^g	52.39 ± 0.13^f	1.87 ± 0.12^b	0.69 ± 0.11^a
myo-Inositol	8.60 ± 0.02^d	5.03 ± 0.01^c	ud	10.58 ± 0.12^f	9.30 ± 0.14^e	0.15 ± 0.01^a	0.70 ± 0.01^b

L5: ethanol precipitate from hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10: sacchachitin (this paper). ud: undetected. MSC: sacchachitin mimic Su et al (1997) from G. lucidum.

DPPH free radical scavenging capability

At a concentration of 10 mg/mL, fractions L6, L7, and L9 revealed higher scavenging bioactivity against DPPH radicals than the other fractions (Figure 3A). However, at 5 mg/mL the fraction L6 only showed a higher activity at 62%, and within 0–5.0 mg/mL, all the other fractions did not show any bioactivity higher than 50%, implicating the polysaccharides in the fruiting bodies of G. lucidum, particularly, the L10 (SGL), are not feasible for scavenge of the organic radicals like DPPH. L10 (SGL) exhibited only 33% of DPPH-scavenging bioactivity even at the highest dosage of 10 mg/mL, conferring an IC₃₃ of 10 mg/mL (the highest scavenging capability never exceeding 33%) (Figure 3A).

Figure 3.  The scavenging capability of different polysaccharide fractions of G. lucidum on DPPH radicals (A), the superoxide anions (B), and the hydroxyl free radicals (C). Values are expressed in means ± SD (n = 3). Fractions L5–L10 are indicated in the flow chart in Figure 1.

Superoxide anion scavenging capability

Nonetheless, in scavenging the water soluble superoxide anions, all the polysaccharide fractions showed activity exceeding 50% at 1.0 mg/mL. The activity of fractions L6, L7, and L9 almost reached 100% at 10 mg/mL. A plateau was soon reached for all fractions at dosages within 1.0–10 mg/mL. Nevertheless, among all factions, the fraction L10 (SGL) exhibited the lowest activity, reaching only 60 and 75% at 1.0 and 10 mg/mL and exhibiting an IC₅₀ of 0.9 mg/mL (Figure 3B).

Hydroxyl free radical scavenging capability

In contrast, fractions L7 and L8 were very active against the hydroxyl free radicals, the activity exceeded 50% at 1.0 mg/mL, attaining 100 and 78% at 5.0 mg/mL, and 100 and 80% at 10.0 mg/mL, respectively (Figure 3C). Fraction L9 revealed a steadily increasing activity in a dose-dependent manner, and fraction L10 (SGL) was moderately active (Figure 3C), only exhibiting an IC₅₀ of 4.8 mg/mL.

To summarize, all the data mentioned above indicate that these water soluble polysaccharides would be more effective in quenching the soluble oxidative species (like the superoxide anions and hydroxyl radicals) than the lipophilic radicals like DPPH.

Comparison of the antioxidative capability between L10 (SGL) and MSC

The L10 sacchachitin usually exhibited higher activity than MSC in scavenging the DPPH radicals (Figure 4A), the superoxide anions (Figure 4B), and the hydroxyl radicals (Figure 4C). Similarly, both preparations did not show an activity >50% against the DPPH radicals (Figure 4A). However, to scavenge the superoxide anions in aqueous system, L10 (SGL) showed activity 59, 70, and 76%, comparing to 43, 60, and 72% for MSC at 1.0, 5.0, and 10.0 mg/mL, respectively (Figure 4B). In contrast, less effect was found for the hydroxyl radical system. At a dosage of 1 mg/mL, neither preparation showed any activity higher than 25%. However, L10 (SGL) exhibited 51 and 55% scavenging bioactivity and MSC showed only 42 and 54% at 5.0 and 10.0 mg/mL, respectively. As cited, the higher the DA, the less the water solubility (Champagne, 2008; Ukai et al., 1983).

Figure 4.  Scavenging capability of the two different sacchachitins isolated by different processes from the fruiting bodies of G. lucidum. For the DPPH radicals (A); for the superoxide anions (B); and for the hydroxyl free radicals (C). Values are expressed in means ± SD (n = 3).

For application, such a biofunctional polymer actually exhibits a limited processibility due to its low solubility. The highly ordered rigid structure of chitin (Supplementary Figure S1) resulting from the formation of intramolecular C₃-OH hydroxyl to C₅-O ring oxygen hydrogen bonds across each β(1→4)-glycosidic linkage restricts chitin units to the low-energy chair conformation, resulting in a rigid and linear polymer backbone (Supplementary Figure S2; Champagne, 2008). This rigidity prevents the polymer’s complete dissolution in common organic solvents (DMSO, DMF, DCM, NMP; Ukai et al., 1983) and even in the aqueous solution (Champagne, 2008).

As mentioned, chitin with a 70% DD is most effective for macrophage activation (Nishimura et al., 1986), similarly found in Table 6. L6 showed the best anti-DPPH radical capability; L7 had the best antihydroxyl free radical capability, and fractions L6, L7, and L9 were best among all in inhibiting the superoxide anions. To compare, the sacchachitin in this paper required an IC₅₀ 1.00 mg/mL for suppressing the syperoxide anions, being far better than 3.30 mg/mL of MSC (Table 6).

Table 6.  Antioxidative IC₅₀ values of different polysaccharide fractions obtained from the fruiting bodies of G. lucidum.

Fraction/sacchachitin	IC50 for DPPH• (mg/mL)	IC50 for •OH (mg/mL)	IC50 for •O2^− (mg/mL)
L5	ud	ud	0.73 ± 0.02
L6	1.99 ± 0.01	ud	0.08 ± 0.01
L7	5.32 ± 0.02	0.50 ± 0.01	0.07 ± 0.01
L8	ud	0.90	0.35 ± 0.02
L9	6.54 ± 0.13	5.14 ± 0.02	0.09 ± 0.01
Sacchachitin (this paper)	ud	4.50 ± 0.04	1.00 ± 0.02
MSC	ud	8.50 ± 0.03	3.30 ± 0.02

L5: Ethanol precipitate of hot water extract; L6: the isoelectric point precipitate from 2% NaOH extract; L7: the three-fold volume ethanol precipitate from 2% NaOH extract; L8: the isoelectric point precipitate from 10% KOH extract; L9: the three-fold volume ethanol precipitate from 10% KOH extract; L10: sacchachitin (this paper). ud: undetected. MSC: sacchachitin mimic Su et al. (1997) from G. lucidum.

In conclusion, the product sacchachitin obtained by our process as fraction L10 from G. lucidum has a hexosamine content 16.3 (% w/w) that is firmly attached to talomannan. The real characteristics of sacchachitin has been evidenced by the FTIR spectral analysis and deacetylation assay. Structurally, it has a hexosamine content 16.3 (% w/w) firmly attached to a talomanna in G. lucidum. This L10 sacchachitin shows better bioactivity than MSC, a product mimic sacchachitin Su et al. (1997) regarding the anti-DPPH, antihydroxyl free radical, and antisuperoxide anion capabilities.

Declaration of interest

The authors declare no conflict of interest.

Online supplementary materials are available at: http://informahealthcare.com/toc/phb