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Keywords:

  • yam tuber mucilage;
  • saliva substitute;
  • xerostomia;
  • elderly

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Objective

To investigate the viscosity of yam tuber mucilage (YTM) and its effects on lysozyme and peroxidase activities in solution phase and on surface phase.

Methods

Two kinds of YTM were extracted, one containing both protein and carbohydrate and the other containing mainly carbohydrate. Hen egg-white lysozyme and bovine lactoperoxidase were used as lysozyme and peroxidase sources, respectively. Viscosity was measured with a cone-and-plate digital viscometer. Lysozyme activity was determined using the turbidimetric method, and peroxidase activity was determined using the NbsSCN assay. Hydroxyapatite beads were used as a solid phase.

Results

The viscosity values of YTM followed a pattern of a non-Newtonian fluid. The carbohydrate concentration affected the viscosity values at all shear rates, while the protein concentration affected the viscosity values at low shear rates. It could be suggested that YTM composed of 1.0 mg/ml protein and 1.0 mg/ml carbohydrate has viscosity values similar to those of unstimulated whole saliva at shear rates present at routine oral functions. Hydroxyapatite-adsorbed YTM significantly increased the adsorption and subsequent enzymatic activities of lysozyme, but not those of peroxidase.

Conclusions

Yam tuber mucilage has viscoelastic properties similar to those of human saliva and enhances the enzymatic activity of lysozyme on hydroxyapatite surfaces.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Xerostomia is one of the most common symptoms in the elderly, with a prevalence of 12–40% in this population[1, 2]. Patients with xerostomia may have complaints that include difficulties in speaking, eating and swallowing. Some patients may also complain of halitosis, chronic oral burning sensation, taste disturbance and intolerance to spicy food. Furthermore, decreased salivary production can lead to oral mucosal infection with Candida, increased risk of dental caries and increased severity of periodontal diseases, which further worsen nutritional problems[3]. Consequently, inadequate saliva production can significantly diminish quality of life[4]. To relieve problems related to xerostomia, most patients use saliva stimulants and/or saliva substitutes. If stimulation of salivary secretion by means of saliva stimulants is ineffective, symptomatic treatment with saliva substitutes may be helpful[3, 5]. Among saliva substitutes, mouthrinse solutions containing sodium carboxymethylcellulose or animal mucins have been extensively used and evaluated[6-8]. Although these saliva substitutes may decrease some symptoms of oral dryness in xerostomic patients, the alleviating effects of today's commercially available substitutes are short-lived and therefore of limited benefit to patients[9, 10].

The development of effective saliva substitutes requires an understanding and mimicry of both the rheological and biological properties of human saliva[10-13]. Therefore, a practical way of developing effective salivary substitutes for xerostomic patients is to identify or develop substances with a viscoelastic pattern similar to that of human whole saliva and to supplement important antimicrobials that restore the decreased antibacterial and antifungal activities in patients with salivary gland hypofunction. In fact, there have been attempts to enhance or restore saliva's own antimicrobial capacity using commercially available oral healthcare products. The antimicrobial host proteins most widely used in such products are lysozyme and lactoperoxidase[14].

Among the salivary proteins, mucus glycoproteins, or mucins, are primarily responsible for the lubricating and film-forming properties of human saliva[15-19]. Therefore, mucins and mucin-like molecules from animal or plant sources are regarded as suitable candidate molecules for saliva substitutes[6, 20-23]. Several studies have reported that saliva substitutes based on animal mucins or mucin-like linseed extracts are more effective than their carboxymethylcellulose-based counterparts[6, 20-22].

Yam (Dioscorea species) is a member of the monocotyledonous family Dioscoreaceae. Fresh tuber slices are widely used as functional foods in Asia, and the dried slices are used as traditional Oriental medicines[24-29]. Recently, we have reported the physical and biological properties of crude yam (the supernatant parts of ground yam samples) and suggested its potential use for the development of saliva substitutes[30]. Results showed that crude yam solutions displayed a viscosity pattern similar to that of human whole saliva. The presence of crude yam affects the enzymatic activity of lysozyme and peroxidase on hydroxyapatite (HA) surfaces, as well as in solution[30].

Yam contains mucin-like molecules exhibiting physical properties that are similar to those of human whole saliva. Yam mucilage is mainly composed of mannan-protein macromolecules[31, 32], which may be used to replace the mucin component of mixed saliva and have the capacity to adhere to the mucosal surface. Therefore, as a further step, information about the physical and biological properties of extracted protein and/or carbohydrate parts of yam mucilage is needed. As for physical properties, we measured the viscosity of extracted yam mucilage. As for biological properties, we examined the effects of extracted yam mucilage on lysozyme and peroxidase activities in solution and on HA surfaces.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Extraction of mucilage from yam tuber

Fresh tubers of yam (Dioscorea batatas) cultivated in Jinju Province of South Korea, which are of a cylindraceous shape with white flesh in a brown peel, were used in these experiments. Extraction of mucilage from yam tuber was performed using two different methods. The first method (method I) was for obtaining extracts containing both protein and carbohydrate[33]. The second method (method II) was for obtaining extracts containing mainly carbohydrate[24].

For method I, washed and peeled yam tubers were cut into strips and ground. After centrifugation at 14 000 g for 30 min, the supernatant was collected and two volumes of isopropanol were added. This solution was stirred quickly and placed at 4°C for 2 h. The precipitates were collected and centrifuged at 14 000 g for 20 min. The final precipitates were washed with two volumes of 95% ethanol and dried at 40°C in an oven.

For method II, washed and peeled tubers were cut into strips. One kilogram of yam tuber was homogenised with 4 l of 50 mM Tris–HCl buffer (pH 8.3) containing 1% vitamin C. After centrifugation at 14 000 g for 30 min, the supernatant was collected and isopropanol was added to a final concentration of 70%. This solution was stirred quickly and placed at 4°C overnight. The precipitates were filtered, dehydrated with 100% isopropanol and then washed with acetone. After drying in an oven at 40°C, the crude yam tuber mucilage (YTM) was ground and collected for further purification using both SDS and heating procedures. One gram of crude YTM powder was dissolved in 200 ml deionised water and kept warm in a water bath at 50°C. Forty millilitres of 5% SDS solution (dissolved in 45% ethanol) was added to the crude YTM solution and kept at 50°C with gentle stirring for 20 min and continuously stirred at room temperature (RT) for another 2 h. Then, this solution was placed in an ice bath to lower the temperature and to precipitate the SDS–protein complex. After centrifugation at 14 000 g for 30 min, the supernatant was collected and isopropanol was added to a final concentration of 70%. This solution was stirred quickly and placed at 4°C for 2 h. The precipitates were filtered, dehydrated with 100% isopropanol, washed with acetone and dried at 40°C in an oven.

Both extraction procedures were performed using the same yam tuber and were also performed six times using different yam tubers. The extracted mucilage was solubilised with simulated salivary buffer (SSB, 0.021 M Na2HPO4/NaH2PO4, pH 7.0, containing 36 mM NaCl and 0.96 mM CaCl2)[34] and used in the experiments.

Determination of total protein and carbohydrate concentrations

Total protein and carbohydrate concentrations were measured in YTM solubilised with SSB. Total protein concentration was determined by the bicinchoninic acid assay using bovine serum albumin as a standard[35]. Total carbohydrate concentration was determined using the phenol/sulphuric acid method with glucose as a standard[36].

A previous report using crude yam solution showed that diluted crude yam solutions at 1:5 and 1:10 in SSB displayed viscosity values similar to those of unstimulated and stimulated whole saliva, respectively. The 1:5 diluted crude yam solution contained approximately 2.0 mg/ml protein and 2.0 mg/ml carbohydrate[30]. Therefore, in the case of extracts obtained using method I, the viscosity measurement was performed at both 1.0 mg/ml (I-1) and 2.0 mg/ml (I-2) protein concentrations. The experiments for enzyme activity were conducted at a 2.0 mg/ml protein concentration. In the case of extracts obtained using method II, the viscosity measurement was performed at both 1.0 mg/ml (II-1) and 2.0 mg/ml (II-2) carbohydrate concentrations. The experiments for enzyme activity were conducted at a 2.0 mg/ml carbohydrate concentration.

Measurement of viscosity

Viscosity measurement was performed with a model LVT Wells–Brookfield cone-and-plate digital viscometer (Brookfield Engineering Laboratories, Stoughton, MA, USA). Shear rates were varied incrementally from low to high speed, and viscosity values were measured at six different shear rates (11.3, 22.5, 45.0, 90.0, 225.0 and 450.0/s). All measurements were taken at 37°C, and 0.5 ml of fluid was used in each test. The viscosity of each sample was measured in triplicate.

Lysozyme and peroxidase

Hen egg-white lysozyme (HEWL) and bovine lactoperoxidase (bLPO) (Sigma-Aldrich, St Louis, MO, USA) dissolved in SSB with phenylmethylsulfonylfluoride (PMSF, final concentrations of 1.0 mM) served as lysozyme and peroxidase sources, respectively. A preliminary experiment showed that PMSF did not affect the enzymatic activity of lysozyme or peroxidase. A concentration of 10 μg/ml HEWL or 12.5 μg/ml bLPO was used for the assays.

Solid phase

Ceramic HA beads (Macro-prep, HA type I) were obtained from Bio-Rad (Hercules, CA, USA). Ten milligrams of HA beads were used in each assay, which provided a surface area of 0.24 m2.

Measurement of lysozyme and peroxidase activities in solution and on HA surfaces

Lysozyme activity was determined using the turbidimetric method[37]. Samples were placed in a lyophilised cell suspension of Micrococcus lysodeikticus ATCC 4698, starting at OD450 = 0.65–0.70, so that the lysozyme present in solution or on the HA surfaces could degrade the bacterial substrate. Peroxidase activity was determined by measuring the rate of oxidation of 5-thio-2-nitrobenzoic acid (Nbs) to 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs)2 by OSCN ions generated during the oxidation of SCN by bLPO[38]. The lysozyme and peroxidase activities were expressed as units/ml in the solution assay and as total units in the surface assay. SSB was used as a blank for the solution samples, while equal amounts of HA beads incubated with SSB were used as a surface blank.

Influence of yam tuber mucilage on lysozyme or peroxidase activity in the solution phase

The effects of YTM on lysozyme activity were examined by incubating 250 μl of the yam solution with 250 μl of HEWL for 10 min at RT. The incubated mixture was placed in a suspension of M. lysodeikticus. The effects of YTM on peroxidase activity were examined by incubating 250 μl of the yam solution with 250 μl of bLPO for 10 min at RT. To 300 μl of reaction mixture for NbsSCN assay, 15 μl of potassium thiocyanate (KSCN, final concentration of 4.2 mM SCN) and 15 μl of sample solution were added, and reaction was initiated by the addition of 15 μl of H2O2 (final concentration of 50 μM). An incubated mixture of buffer with either HEWL (or bLPO in the case of peroxidase activity) was used as a control. For the blank reaction, an incubated mixture of yam solution with buffer or an incubated buffer alone was used. All experiments were performed in duplicate.

Influence of yam tuber mucilage on lysozyme or peroxidase activity on the surface phase

To determine the influence of YTM on the enzymatic activity of lysozyme or peroxidase adsorbed to a HA surface, two different experiments were performed: (i) the effects of adsorbed YTM on subsequent adsorption of lysozyme or peroxidase and (ii) HA-absorbed lysozyme or peroxidase activity after pre-incubation with YTM.

First, to examine the effects of YTM on the adsorption or subsequent activity of lysozyme or peroxidase when present on the HA surface, 10 mg of HA beads was coated with 300 μl of the yam solution for 30 min at RT. After coating, the beads were washed five times with SSB. The yam-coated HA beads were then incubated with 300 μl of HEWL (or bLPO) for 30 min at RT. Unbound HEWL (or bLPO) molecules were removed through five SSB washes. The beads were then used for lysozyme or peroxidase assays, as described above. The enzymatic activities of these samples were compared with those of the bare HA sample surfaces coated with HEWL (or bLPO).

Second, to examine the effects of pre-incubation of YTM with lysozyme or peroxidase on the adsorption or subsequent activity of lysozyme or peroxidase on HA surface, 300 μl of the yam solution was incubated with 300 μl of HEWL (or bLPO) solution for 10 min at RT. HA beads were incubated with 600 μl of the mixture for 30 min at RT and then washed five times with SSB to remove unbound molecules. The enzymatic activities of these samples were compared with those of the HA samples coated with the pre-incubated mixture of HEWL (or bLPO) with buffer. Equal amounts of HA beads incubated with SSB were used as blanks in all experiments. All experiments were performed in duplicate.

Statistics

The Wilcoxon's signed rank test or Mann–Whitney U test was used to compare the mean values of variables. P-values <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

Extraction of mucilage from yam tuber

The amounts of YTM obtained from 100 g of yam tuber using methods I and II were 0.53 ± 0.18 g and 0.16 ± 0.09 g, respectively. The protein–carbohydrate ratio of YTM obtained using method I was 1:0.55. The protein amount of YTM obtained using method II was negligible.

Viscosity

Figure 1 shows the viscosity values of YTM extracted via methods I and II at six different shear rates: I-1, at 1.0 mg/ml protein and 0.55 mg/ml carbohydrate; I-2, at 2.0 mg/ml protein and 1.10 mg/ml carbohydrate; II-1, at 1.0 mg/ml carbohydrate; II-2, at 2.0 mg/ml carbohydrate. The viscosity values of YTM dissolved in SSB followed a pattern of a non-Newtonian fluid, and the viscosity values increased with increasing protein or carbohydrate concentration, as expected. The viscosity values of II-1 or II-2 were higher than those of I-1 or I-2, respectively. When the concentration of carbohydrate was increased from 1.0 to 2.0 mg/ml (between II-1 and II-2), the viscosity values were significantly increased at all six shear rates (< 0.05). When the protein concentration was increased from 0 to 2.0 mg/ml (between I-2 and II-1), the viscosity values were increased more at lower shear rates, and a significant difference was found at a shear rate of 11.3/s (< 0.05).

image

Figure 1. Viscosity values of yam tuber mucilage extracted using method I or II. I-1, at 1.0 mg/ml protein and 0.55 mg/ml carbohydrate; I-2, at 2.0 mg/ml protein and 1.10 mg/ml carbohydrate; II-1, at 1.0 mg/ml carbohydrate; II-2, at 2.0 mg/ml carbohydrate.

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Figure 2 shows the viscosity values of YTM compared with those of human saliva from our previous study[13]. The viscosity values of I-1 were very close to those of stimulated whole saliva at shear rates of 45.0 and 90.0/s. When the concentration of YTM was adjusted to 1.0 mg/ml protein and 1.0 mg/ml carbohydrate (III) through the addition of extract obtained using method II to the extract obtained using method I, the viscosity values were very close to those of unstimulated whole saliva at shear rates of 45.0 and 90.0/s.

image

Figure 2. Viscosity values of yam tuber mucilage compared with those of human saliva. I-1, at 1.0 mg/ml protein and 0.55 mg/ml carbohydrate; III, at 1.0 mg/ml protein and 1.0 mg/ml carbohydrate; UWS, unstimulated whole saliva; SWS, stimulated whole saliva.

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Influence of yam tuber mucilage on lysozyme or peroxidase activity in the solution phase

The YTM extracted using both methods did not affect the enzymatic activities of HEWL in the solution phase. Although the enzymatic activities of bLPO were increased by the YTM (15.2 ± 27.5% in method I, 22.8 ± 20.9% in method II), there were no significant differences between bLPO only and the mixture of bLPO and YTM in the solution phase (Table 1).

Table 1. Influence of yam tuber mucilage on lysozyme and peroxidase activities in the solution phase
 Yam tuber mucilage extracted using method IYam tuber mucilage extracted using method II
  1. Statistical significance was evaluated using the Wilcoxon's signed rank test.

  2. HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase.

N = 6HEWLHEWL + YamSignificanceHEWLHEWL + YamSignificance
Lysozyme activity (Units/ml)378.8 ± 35.7356.5 ± 61.6= 0.345359.0 ± 88.2356.2 ± 118.0= 0.833
N = 6bLPObLPO + YamSignificancebLPObLPO + YamSignificance
Peroxidase activity (mUnits/ml)2.617 ± 0.9303.031 ± 1.164= 0.1732.176 ± 0.7612.704 ± 1.118= 0.074

Influence of yam tuber mucilage on lysozyme or peroxidase activity on the surface phase

  • 1.
    Effects of adsorbed YTM on lysozyme and peroxidase activities

The adsorbed YTM significantly enhanced the adsorption and subsequent enzymatic activities of HEWL (< 0.05), but not those of bLPO on the surfaces of HA beads. These effects were the same for samples obtained via methods I and II (Table 2).

Table 2. Effects of hydroxyapatite-adsorbed yam tuber mucilage on lysozyme and peroxidase activities
 Yam tuber mucilage extracted using method IYam tuber mucilage extracted using method II
  1. Statistical significance was evaluated using the Wilcoxon's signed rank test.

  2. HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase.

N = 6HEWLHEWL + YamSignificanceHEWLHEWL + YamSignificance
Lysozyme activity (Units)5.0 ± 2.333.8 ± 9.2= 0.0284.7 ± 3.317.2 ± 11.7= 0.028
N = 6bLPObLPO + YamSignificancebLPObLPO + YamSignificance
Peroxidase activity (mUnits)0.064 ± 0.0110.059 ± 0.012= 0.1160.058 ± 0.0090.065 ± 0.005= 0.116
  • 2.
    Effect of pre-incubation of YTM with lysozyme (or peroxidase) on surface lysozyme (or peroxidase) activity

When HA beads were exposed to the pre-incubated mixture of YTM and HEWL, the enzymatic activities of immobilised HEWL were greatly increased (< 0.05). In the case of peroxidase activity, the enzymatic activities of immobilised bLPO on HA beads were not increased. These effects were the same for samples obtained via methods I and II (Table 3).

Table 3. Effects of pre-incubation of yam tuber mucilage with lysozyme or peroxidase on the subsequent lysozyme or peroxidase activity on a hydroxyapatite surface, respectively
 Yam tuber mucilage extracted using method IYam tuber mucilage extracted using method II
  1. Statistical significance was evaluated using the Wilcoxon's signed rank test.

  2. HEWL, hen egg-white lysozyme; bLPO, bovine lactoperoxidase.

N = 6HEWLHEWL + YamSignificanceHEWLHEWL + YamSignificance
Lysozyme activity (Units)0.9 ± 0.412.9 ± 6.8= 0.0281.4 ± 0.68.9 ± 8.7= 0.028
N = 6bLPObLPO + YamSignificancebLPObLPO + YamSignificance
Peroxidase activity (mUnits)0.033 ± 0.0120.026 ± 0.011= 0.1720.027 ± 0.0040.028 ± 0.017= 0.463

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

The practical way of developing effective salivary substitutes for xerostomic patients is to obtain candidate substances with a viscoelastic pattern similar to that of human whole saliva and add supplements to restore the decreased antimicrobial activities in patients with dry mouth. The results of the present study indicated that YTM can be a good candidate for an effective saliva substitute, considering its physical and biological properties. It has been suggested that the mannan-protein macromolecules of YTM might replace human salivary mucins and that the sugar moieties could form a surface layer of water that protects the oral mucosa from friction[30] and prevents oral dryness by maintaining mucosal wetness[39].

The results of the present study showed that protein, as well as carbohydrate components, are essential to the maintenance of viscoelastic properties of YTM. Carbohydrates are important to maintain a certain level of viscosity at both low and high shear rates, while proteins are important to maintain a certain level of viscosity at low shear rates. When the results of the present study were compared with the viscosity values of human whole saliva[13] at shear rates that would exist during oral functions such as swallowing or speech (from 60 to 160/s)[40], the viscosity values of the I-1 sample (extracts containing protein 1.0 mg/ml, carbohydrate 0.55 mg/ml) were similar to those of stimulated whole saliva. The results also suggested that YTM having 1.0 mg/ml protein and 1.0 mg/ml carbohydrate display viscosity values similar to those of unstimulated whole saliva. The viscosity values of extracted YTM were lower than those of crude yam solution at the same concentration of protein or carbohydrate[30]. This may be due to the modification of macromolecules during the extraction procedures, which subsequently also affects the interactions between macromolecules.

We also found that the patterns of YTM influence on the enzymatic activity of lysozyme or peroxidase were different in solution and on HA surfaces. Of the antimicrobial molecules identified in saliva and salivary pellicles on HA surfaces, lysozyme and peroxidase are prominent antibacterial components widely distributed in various biological fluids including saliva, tears, milk and cervical secretions[41, 42]. Moreover, these antimicrobials, either alone or in combination with other antimicrobial molecules, have been incorporated in commercial oral healthcare products to restore the antimicrobial capacity of saliva[14]. Therefore, YTM in saliva substitutes, host-derived antimicrobial salivary molecules and supplemented antimicrobials may exist simultaneously in whole saliva and tooth pellicles of patients with salivary hypofunction, and interactions between these molecules may occur. Such interactions may modify the antimicrobial activity of the innate defence molecules in distinct ways in solution phase or on surface phase[43, 44].

The YTM did not affect the enzymatic activities of HEWL or bLPO in the solution phase, which was consistent with the results using crude yam[30]. The enhancement of lysozyme activity in conjunction with YTM adsorbed onto a surface appears to be due to an increased amount of lysozyme adsorption, as compared with bare HA surfaces. The increase in enzymatic activity on HA surfaces after exposure to a mixture of pre-incubated YTM and HEWL could be explained by the same reasoning. These effects did not occur in the case of bLPO, which differed from the results using crude yam[30]. It could also be suggested that extraction procedures may modify the macromolecules or the interactions between macromolecules. In fact, the direct binding of peroxidase to mannans has been suggested[45], and modification of macromolecule structure during extraction might affect interactions between such molecules.

Because the in vitro environment does not completely mirror what occurs in the mouth in vivo, there are several issues to be considered before the results of the present study can be extrapolated to an in vivo situation. This difference may be due in part to surface differences between tooth mineral and synthetic HA beads. Other possibilities are the presence of proteolytic activities in the oral environment. The influences of YTM on pellicle formation and on bacterial adherence to the enamel or restorations should also be studied. In addition, there are other aspects to be considered when developing more effective saliva substitutes. It has been suggested that the formulation and delivery method of saliva substitutes affect the therapeutic efficacy of the substitutes and composition of oral microflora in patients with dry mouth[46, 47].

In conclusion, the present study provides an objective observation on the possibility of YTM for potential use in the development of effective saliva substitutes. YTM displayed a viscoelastic property similar to that of human whole saliva. The presence of YTM increased the enzymatic activity of lysozyme on HA surfaces.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References

This work was supported by the National Research Foundation of Korea Grant through the Oromaxillofacial Dysfunction Research Center for the Elderly (No. 2011-0028230) at Seoul National University in Korea.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conflict of Interest
  8. Acknowledgements
  9. References
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