Structural Characteristics and Antioxidative Capability of the Soluble Polysaccharides Present in Dictyophora indusiata (Vent. Ex Pers.) Fish Phallaceae

Dictyophora indusiata (Vent. ex Pers.) Fish Phallaceae (Chinese name Zhu-Sūn, the bamboo fungi) has been used as a medicinal mushroom to treat many inflammatory, gastric and neural diseases since 618 AD in China. We hypothesize that the soluble polysaccharides (SP) present in D. indusiata and their monosaccharide profiles can act as an important role affecting the antioxidative capability, which in turn would influence the biological activity involving anti-inflammatory, immune enhancing and anticancer. We obtained six SP fractions and designated them as D1, a galactoglucan; D2, a galactan; D3, the isoelectrically precipitated riboglucan from 2% NaOH; D4, a myoinositol; D5 and D6, the mannogalactans. The total SP accounted for 37.44% w/w, their molecular weight (MW) ranged within 801–4656 kDa. D3, having the smallest MW 801 kDa, exhibited the most potent scavenging effect against the α,α-diphenyl-β-picrylhydrazyl, •OH−, and •O2 − radicals, yielding IC50 values 0.11, 1.02 and 0.64 mg mL−1, respectively. Thus we have confirmed our hypothesis that the bioactivity of D. indusiata is related in majority, if not entirely, to its soluble polysaccharide type regarding the MW and monosaccharide profiles.


Introduction
Dictyophora indusiata (Vent. Ex Pers.) Fish Phallaceae (Chinese name Zhu Sūn, meaning the bamboo mushroom), synonymously called Phallus indusiatus, is frequently used under the name Veiled lady mushroom ( Figure 1). The folkloric consumption of D. indusiata in the ancient China began around 618 AD, which pointed mainly to the nutritional bioactivities, like benefits to eyes and tonics to cardiovascular systems; and partially to the medicinal effect like mental tranquilization, antitumor, and tonics, and so forth, [1][2][3][4]. It consists of seven essential amino acids, 12 precious metallic ions and rather high content of vitamin E [5,6], β-carotene, thiamine, riboflavin, nicotinic acid, l-ascorbic acid, calcium and phosphate [7,8].
A review by Lindequist indicted that many edible mushrooms exhibit a broad spectrum of bioactivities that are beneficially acting as a complementary alternative medicine (CAM) [9]. As often cited, the therapeutic efficacy of mushrooms includes antibacterial, antifungal, anti-multiresistant bacterial, antiviral, immunomodulating, antitumor or antitumor adjuvant, cytostatic, immunosuppressive and antiallergic, antioxidative and anti-inflammatory, antiatherosclerogenic, hypoglycemic, hepatoprotective, and neurotrophic functions [9].
As is well known, the antioxidative capability may be relevantly related with its immune-enhacing, anti-inflammatory and anticancer bioactivities. We hypothesize that the SP present in D. indusiata and their monosaccharide profiles can Evidence-Based Complementary and Alternative Medicine 3 act as an important role affecting the antioxidative capability, which in turn would influence the biological activity as mentioned ( Figure 2).

Proximate Compositional Analysis.
The proximate analysis was performed according to the methods described in AOAC [33].

Fractionation of Soluble
Polysaccharides. The fractionation of different SP from the fruiting bodies was performed according to the flowchart shown in Figure 3 [34].

The Soluble Polysaccharide Fraction D1.
Briefly, desiccated fruiting bodies of D. indusiata (100 g) (DS) was extracted three times while avoiding direct sunlight by refluxing with 2000 mL of double distilled water at 90 • C with constant stirring at 400 × g for 2 h. On cooling, the solution was centrifuged at 10 000 × g for 30 min. The supernatant (S1) was collected. The extraction was repeated three times. The residue (R1) was kept for further treatment. The supernatants were combined and acidified with 1 N HCl to pH 4. In case that the precipitation should occur instantaneously, centrifuge the solution immediately at 10 000 × g to remove the first precipitate. Otherwise, to the solution (S2) a 3fold volume of ethanol was added, stirred for 30 min, left to stand at ambient temperature for 1 h and centrifuged at 10 000 × g for 30 min. The precipitate (ppt-1) occurred in solvent ethanol/hot water (3 : 1) was collected, dialyzed at 4 • C for 3 days, lyophilized and weighed (SP D1) ( Figure 2).

The Soluble Polysaccharide Fraction D2.
To the residue (R1) 2000 mL of 0.04 N HCl were added and mixed well. The mixture was refluxed while avoiding direct light sunlight at 80 • C for 2 h. The extraction was repeated three times, combined, and centrifuged at 10 000 × g for 30 min to collect the precipitate (R2). To the supernatant (S4) a 3-fold volume of ethanol was added, stirred at room temperature, left to stand for 1 h and centrifuged at 10 000 × g for 30 min to collect the precipitate (ppt-2). The supernatant (S5) was discarded. The ppt-2 obtained in the mixed solvent ethanol/0.04N HCl (3 : 1) was dialyzed at 4 • C, lyophilized and weighed to obtain the SP D2 ( Figure 2).

The Soluble Polysaccharide Fraction D5.
To the residue R2 2000 mL of 2% NaOH were added and mixed well. The mixture was refluxed while avoiding direct sunlight at 80 • C for 2 h. The extraction was repeated for three times. The extracts were combined, cooled, and subjected to centrifugation at 10 000 × g for 30 min. The precipitate (ppt-5) was discarded ( Figure 2). To the supernatant (S10) 1 N HCl was added to adjust the pH to 4.0. The acidified solution was left to stand at ambient temperature for 1 h. The mixture was centrifuged at 10 000 × g to collect the isoelectric precipitate from 10% KOH (ppt-6), which was dialyzed, lyophilized and weighed to obtain the SP D5 ( Figure 2).

The Soluble Polysaccharide Fraction D6.
To the supernatant (S11) 3-fold volume of ethanol was added. The solution was stirred at ambient temperature for 30 min and centrifuged at 10 000 × g. The precipitate obtained in the ethanol/10% KOH (3 : 1) mixed solvent was collected, lyophilized and weighed to obtain the SP D6 ( Figure 2).

The Soluble Polysaccharide Fraction D3.
Alternatively, to the supernatant S6 2000 mL of NaOH was added and refluxed at 80 • C for 2 h. The mixture was left to stand at ambient temperature for 1 h and centrifuged at 10 000 × g to obtain the supernatant (S7). The precipitate (ppt-3) was discarded ( Figure 2). An amount of 1 N HCl was used to adjust the pH value of S7 to 4.0. The acidified solution was left to stand at ambient temperature for 1 h and centrifuged at 10 000 × g for 30 min. The precipitate occurred by the isoelectric precipitation from 2% NaOH (ppt-4) was collected, dialyzed, lyophilized and weighed to obtain the SP D3 ( Figure 2).

The Soluble Polysaccharide Fraction D4.
To the supernatant (S8) a 3-fold volume of ethanol was added and stirred for 30 min. The solution was centrifuged at 10 000 × g for 30 min to obtain the precipitate. The supernatant (S9) was discarded ( Figure 2). The precipitate collected from the ethanol/2% NaOH (3 : 1) was dialyzed, lyophilized and weighed to obtain the SP D4 ( Figure 2).  to make a final volume of 5 mL. The mixture was thoroughly agitated to facilitate the dissolution and centrifuged at 2500 × g 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% NaN 3 at a flow rate 0.5 mL min −1 on the Sephadex G-100 column (internal diameter (i.d.) × l = 2.5 × 100 cm). The fraction collector (ISCO Retriever 500, Isco., Lincoln, NE) was used to collect the eluents, 6 mL per tube. The collection was stopped at the 100th tube. The calibration curve was established using the authentic dextrans having molecular masses 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 [36].

Analysis for Ratio Carbohydrate to Peptide Moiety.
Different SP (D1-D6) was subjected to the Sephadex G-100 column separation. The Dubois' phenol-sulfuric acid colorimetric method [30] was followed to determine the carbohydrate content. Briefly, to each milliliter of the polysaccharide fraction 1 mL of phenol reagent (5%) and 5 mL of sulfuric acid were added. On cooling to the ambient temperature, the optical density was read at 490 nm. Alternatively, Coconnier et al. [37] was followed for the determination of peptidomoiety. The absorbance at 280 nm was taken straightforwardly for the six SP fractions (D1-D6) obtained from GPC.

Acid Hydrolysis.
Three grams of each sample SP were accurately weighed and transferred separately into a 2 mL reaction vessel, to which 2 mL of 6 M HCl was added. The dissolved oxygen was purged off by nitrogen blowing for Evidence-Based Complementary and Alternative Medicine 5 10 min. The vessel was sealed, placed in the derivatization reactor, and heated at 110 • C for 24 h until the peptide moiety was completely hydrolyzed. The hydrolyzed product was lyophilized. The desiccated product was re-dissolved in 0.3 mL of 0.01 M HCl to obtain the sample amino-acid mixture (AM).

Derivatization of Amino Acid and
Extraction. An amount of 0.3 mL of authentic and AM (0.6 mL) solutions were respectively placed into a 3-mL reaction vessels, to which the internal standard solution of norleucine (0.01 mL, 10 mg mL −1 ) was added. When vigorously agitated, 0.1 mL of ethyl chloroformate and 1 mL of alcohol-pyridine were added and mixed thoroughly. To the mixture 2 mL of chloroform were added. When mixed well for 1 min to facilitate the derivatization and the extraction, water (0.7 mL) was added, shaken well, and left to stand for 5 min to facilitate phase separation. The supernatant was discarded. The lower layer (i.e., the chloroform layer) was transferred into a new tube and dehydrated with 0.1 g of anhydrous sodium sulfate. The dehydrated chloroform extract was transferred into the sample vessel and analyzed by GC/MS. The temperature of the detector FID and the injection port was held at 305 and 300 • C, respectively. The elution temperature was programmed initially at 50 • C for 1 min, then raised at an elevation rate 10 • C min −1 to 300 • C and held at which for 6.5 min.

Assay for Antioxidative Capability-Preparation of SP Solutions.
To 250 mg of SP (D1-D6) samples, 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 × g for 10 min to remove the insoluble precipitate. The supernatant was collected (SP stock solution) for the following assays.

Scavenging Capability for DPPH Radicals.
Method of Blois [38], later modified by Shimada et al. [39], was followed for assay of the DPPH radicals. Briefly, to 1 mL of DPPH (methanolic DMSO solution, 0.5 mM, Sigma-Aldrich Co., MO, USA) an appropriate amount of each SP stock solution (20-100 μL) or the standard was added. The mixture was thoroughly agitated and left to stand for 30 min. The absorbance was read at 517 nm using the BioMate 5 spectrophotometer (Thermo Electron Corporation, San Jose, CA, USA). DHA was used as the reference standard. The percent DPPH radical scavenging activity was calculated as directed [38,39].

Scavenging Capability for Hydroxyl-and Superoxide
Anion Radicals. The modified method of Adom and Liu 2.14. Statistical Analysis. Data obtained in the same group were analyzed by Student's t-test with computer statistical software SPSS 10.0 (SPSS, Chicago, IL). Statistical analysis system software was used to analyze the variances. Duncan's multiple range tests were used to test their significances of difference between paired means. Significance of difference was judged by a confidence level of P < .05.

Monosaccharide
Composition. Ten kinds of monosaccharides were found in the SP of D. indusiata. Obeying the conventional naming rule, fraction D1 was designated glucogalactan; D3, the riboglucan, and both D5 and D6, the mannogalactans. Interestingly, huge amount of myo-inositol was simultaneously found in fractions D4, D2), and D1 (Table 3).

Discussion
The fruiting bodies of D. indusiata contained huge amount of carbohydrates (46.89%). In parallel, its crude protein content was only 6.07%, implicating the possible peptidoglycan structure underling the main medicinal effect. Data revealed that only fractions D1, D2, D4 and D6 were peptidoglycan in nature. As a contrast, fractions D3 and D5 were typically characteristic of glycoproteins. Moreover, the high total SP content (37.44%) may also implicate an alternate therapeutic use. By insight of monosaccharide profile, fraction D1 was designated a glucogalactan; D3, a riboglucan; D5 and D6, mannogalactans. Fraction D4, having myo-inositol content 92.5%, almost was approaching the pure myo-inositol. Lin purified a polysaccharide xylose-containing Di-S2P from D. indusiata fruiting bodies. Di-S2P had MW 870 kDa [19], however xylose was undetected in D3. Worth noting, D4 and D6 uniquely contained allose ( Table 3). As well known, inositol epimerase governs the conversion of myo-inositol to chiro-inositol [42][43][44], an insulin dependent process related with immunological activity. Myoinositol is an essential nutrient required for keratinocytes growth [45,46]. The actual role of such an unusually high content of myo-inositol in D. indusiata acting as immunomodulator remains to further investigation. D3 possessed the most potent DPPH radical scavenging capability prevailing over the mannogalactan (D6), implicating the complicate phytochemical-therapeutics relationship [47]. As well cited, the binding of glucans having (1 → 3)-β-dglucosan main frame with β-d-(1 → 6) branches onto the cell surface of cytotoxic macrophages, helper T and NK cells will trigger immunopotentiation [47,48]. The degree of branching (DB) between 0.20 and 0.33 and the triple helical structures are important for immunopotentiating [47]. Conversely, some insoluble aggregates also had been reported to be more stimulatory than the soluble polymers Values are expressed in means ± SD (n = 3). [47]. A water-insoluble, alkali-soluble extracellular glucan (CO-1) (average MW ∼632 kDa) isolated from Cordyceps ophioglossoides was shown to inhibit the Sarcoma 180 tumor [48].
To conclude, the fruiting bodies of D. indusiata contain huge amount of SP (37.5%) that exhibits MW ranging within 801-4656 kDa. Among which, the fraction D3 (the isoelectrically precipitated riboglucan from 2% NaOH) has the smallest MW 801 and is the most potent SP regarding the antioxidative capability. Furthermore, the huge amount of myo-inositol present can be relevantly associated with its additional immunobioactivity. Thus, we have confirmed our hypothesis that the bioactivity of D. indusiata is related in majority, if not entirely, to its soluble polysaccharides.