Physicochemical properties and biological activities of Thai plant mucilages for artificial saliva preparation

Abstract

Context: Plant mucilages can be found in various parts of several Thai plants, which can be used as thickening, moisturizing, and lubricating agents in artificial saliva formulations.

Objective: The objective of this study was to evaluate the physicochemical properties, biological activity, and cytotoxicity of Thai plant mucilages.

Materials and methods: The mucilages from Thai plants were extracted by various processes (temperature and pH variation, microwave oven, steam, and Tris-HCl buffer extraction). The viscosity and the rheology were evaluated using viscometer. Antioxidative activities including DPPH radical scavenging and metal chelating activities were investigated. The mucilages were determined for cytotoxicity on normal human gingival fibroblasts and anti-adherent activity of Streptococcus mutans.

Results: Mucilages from Ocimum citriodorum Vis. (Lamiaceae), Artocarpus heterophyllus Lam. (Moraceae), Abelmoschus esculentus (Linn.) Moench. (Malvaceae), and Basella alba Linn. (Basellaceae) exhibited pseudoplastic non-Newtonian rheology. The highest DPPH radical-scavenging and metal-chelating activities were observed in the mucilages from B. alba (microwave, 3 min) and A. esculentus (microwave, 1 min) with the SC₅₀ and MC₅₀ values (50% of scavenging activity and 50% of metal chelating activity, respectively) of 0.71 ± 0.32 and 1.11 ± 0.52 mg/ml, respectively. Most mucilages exhibited no cytotoxicity to normal human gingival fibroblasts. The mucilage from A. esculentus (microwave, 5 min) gave the shortest wetting time of 2.75 ± 0.51 min. The highest S. mutans adhesion inhibition was observed in A. esculentus (pH 11) of 5.39 ± 9.70%.

Discussion and conclusion: This study has indicated the suitable physicochemical and biological properties and the potential application of mucilages from Thai plants for artificial saliva preparation.

• Adhesion inhibition
• antioxidant
• cytotoxicity
• pseudoplastic non-Newtonian rheology
• wetting time

Introduction

Polysaccharide hydrocolloids including mucilages, gums, and glucans are abundant in nature and commonly found in many higher plants. These polysaccharides constitute a structurally diverse class of biological macromolecules with a broad range of physicochemical properties. Polysaccharides are widely used for various applications in pharmacy and medicines. Mucilages are most commonly used as adjuvant in pharmaceutical preparations such as thickening, binding, disintegrating, suspending, emulsifying, stabilizing, and gelling agents and in drug delivery systems as sustained and controlled release formulations of several drugs (Deore & Khadabadi, 2008). Saliva is one of the most complex but versatile and important body fluids. It contains a number of systems which serve a wide spectrum of physiological needs. Saliva is required to swallow food, speak, and protect the oral mucosa and the teeth from infection. This fluid contains a variety of electrolytes, peptides, glycoproteins, and lipids. The normal human saliva demonstrated a non-Newtonian, pseudoplastic behavior with the average viscosity of 9.13 cP (Preetha & Banerjee, 2005). Pseudoplastic non-Newtonian or shear-thining fluids have lower apparent viscosity at higher shear rates, and are usually dispersion of large, polymeric molecules in proper solvents (Hamedi & Rahimian, 2011). A loss or reduction of saliva can result in significant problems such as carries, periodontal diseases, difficulties with denture wearing, eating, talking, altered taste sensation, as well as higher risk of candidiasis and mucositis resulting in an overall reduction in the quality of life. Sreebny (1988) has defined xerostomia as “the subjective feeling of oral dryness”, resulted from salivary gland hypofunction. The prevalence of xerostomia varies from 13 to 28% in elder populations and increases up to 60% in patients living in long-term care facilities (Ettinger, 1996). Treatment of the patients with reduced saliva can be by using artificial saliva. Usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC), sodium carboxymethylcellulose (SCMC), and hydroxyethylcellulose as thickening and lubricating agents. As known, plant mucilages can be found in various parts of several plants, which can be used as thickening, moisturizing, and lubricating agents in artificial saliva formulations. Examples of Thai plants which have been reported to contain mucilages are Basella alba Linn., Hibiscus esculentus Linn., Litsea glutinosa (Lour.) C.B. Robinson, Ocimum canum Sims., Plantago ovate Forssk., Scaphium scaphigerum G. Don., and Trigonella foenum-graecum Linn. (Palanuvej et al., 2009). Plant mucilages are composed of polysaccharides, resins, or tannins (Avachat et al., 2011). In this study, the physicochemical properties, biological activity, and cytotoxicity of the plant mucilages were evaluated. The plant mucilages which exhibited the most characteristics similar to the normal human saliva will be selected for the further developed as artificial saliva formulations.

Materials and methods

Materials

Twenty-three plants including Abelmoschus esculentus (Linn.) Moench (Malvaceae), Aloe barbadensis Mill. (Lilaceae), Artocarpus heterophyllus Lam., Asparagus racemosus Wild. (Moraceae), Basella alba Linn. (Basellaceae), Citrullus lanatus (Thunb.) Mats. & Nakai. (Cucurbitaceae), Cucumis sativus Linn. (Cucurbitaceae), Cucurbita moschata Decne. (Cucurbitaceae), Dimocarpus longan Lour. (Sapindaceae), Hylocereus undulates (Cactaceae), Ipomoea batatas (L.) Poir. (Convolvulaceae), Litchi chinensis Sonn. (Sapindaceae), Luffa cylindrical (Linn.) Roem (Cucurbitaceae), Lycopersicon esculentum Mill. (Solanaceae), Manilkara achras Fosberg. (Sapotaceae), Moringa oleifera Lam. (Moringaceae), Musa sapientum Linn. (Musaceae), Ocimum citriodorum Vis. (Lamiaceae), Passiflora edulis Sims. (Passifloraceae), Solanum melongena L. (I) (Solanaceae), Solanum melongena L. (II) (Solanaceae), Tamarindus indica Linn. (Fabaceae), and Vigna radiata (L.) R. Wilcz. (Fabaceae) were purchased from local fresh market in Chiang Mai province, Thailand, and identified by a botanist (Ms. Suda Saowakon) and the voucher specimens were deposited at Natural Products Research and Development Center (NPRDC), Science and Technology Research Institute (STRI), Chiang Mai University in Thailand. Tris-HCl, hydrochloric acid, sodium hydroxide, vitamin C (l-(+)-ascorbic acid) and 2,2-diphenyl-1-picryhydrazyl (DPPH) were from Sigma Chemical Co. (St. Louis, MO). All reagents were of analytical grade.

Extraction of plant mucilages

The plant was cut into small pieces, added with distilled water (seven times of the plant weight) at 25 and 60 °C, and homogenized in a blender for 1 min. The extract was pressed through the muslin cloth. The filtrate was centrifuged at 4660g, 25 °C for 30 min and the supernatant was collected. The supernatant was then mixed with ethanol at the ratio of the extract to ethanol of 1:3 v/v. The mixture was then centrifuged at 4660g, 25 °C for 15 min. The sediment was collected and the remaining ethanol was evaporated by a rotary evaporator (Buchi, Flawil, Switzerland). The mucilage was lyophilized and kept at room temperature (25 ± 2 °C) until used.

Optimization of mucilage extraction

The plant mucilages which demonstrated the pseudoplastic non-Newtonian flow were selected to develop the extraction processes as follows:

Temperature and pH variation extraction: The plant was cut into small pieces and homogenized with distilled water (seven times of its weight) at various temperatures (25, 60, and 100 °C) and pH (pH 2 and 11) in a blender for 1 min. The extract was then pressed through the muslin cloth. The filtrate containing mucilage was centrifuged at 4660g, 25 °C for 30 min and the supernatant was collected.

Microwave oven extraction: The plant in small pieces was macerated in distilled water (seven times of its weight) for 24 h and microwaved at 600 W intensity for 1, 3, or 5 min. The extract was then pressed through the muslin cloth. The filtrate containing mucilage was centrifuged at 4660g, 25 °C for 30 min and the supernatant was collected.

Steam extraction: The plant in small pieces was cooked by steam for 30 min. The steamed sample was then homogenized in a blender for 1 min and pressed through the muslin cloth. The filtrate containing mucilage was centrifuged at 4660g, 25 °C for 30 min and the supernatant was collected.

Tris-HCl buffer extraction: The plant in small pieces was mixed with Tris-HCl buffer, pH 8.3 (seven times of its weight) for 1 min. The extract was then pressed through the muslin cloth. The filtrate containing mucilage was centrifuged at 4660g, 25 °C for 30 min and the supernatant was collected.

The supernatant containing the mucilage was precipitated by ethanol as previously described in the section “Extraction of plant mucilages”.

Determination of viscosity and rheology

The lyophilized mucilage was reconstituted in distilled water at 1% w/v and homogenized for 15 min. The viscosity and the rheology of the mucilage were evaluated using a viscometer (Myr VR 3000 model, Tarragona, Spain) with the shear rate range of 5–200 rpm. The shear stress of the mucilage was calculated by the following equation:

The concentration of the mucilages was varied to obtain the viscosity of 9 cP (similar to the average viscosity of human saliva). SVEU of the mucilage was calculated as followed:

Sodium carboxymethylcellulose (SCMC), a viscosity modifying agent, was used as a standard. The 9 cP viscosity of SCMC at 0.2%w/v gave the pseudoplastic non-Newtonian flow (Banerjee & Das, 1998). The mucilages that exhibited the pseudoplastic non-Newtonian flow (the flow of human saliva) were selected to determine phytochemical constituents and antioxidant activities including DPPH free radical scavenging and metal chelating activities.

Phytochemical assays

The mucilages which exhibited the pseudoplastic non-Newtonian flow behavior were analyzed for phytochemical constituents (anthraquinones, glycosides, tannins, carotenoids, flavonoids, and alkaloids) using the standard methods (Manosroi et al., 2010). For anthraquinones, 0.05 g of the mucilage was put into a dry test tube, 2 ml of chloroform was added and shaken for 5 min. The mucilage was filtered. The filtrate was mixed with an equal volume of 10% ammonia solution and shaken. A pink violet or red color in the ammoniacal layer (lower layer) indicated the presence of anthraquinone. The qualitative assay of reducing sugars was performed by the TLC method. The mucilage dissolved in water was spotted on the silica gel plate in comparing with the standard reducing sugars (glucose, fructose, and sucrose). The filtrate was resolved on the TLC plate coated with silica gel 60. The mobile phase was butanol/acetic acid/diethyl ether/water (9:6:1:3). The spot on the plate was sprayed with 10% H₂SO₄ and heated. Sucrose, glucose, and fructose were used as the standards. For tannins, 0.05 g of the mucilage was mixed with 2 ml of 15% FeCl₃ solution. The blue-black precipitate indicated the presence of tannins. For carotenoids, each mucilage sample was extracted with chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 0.1 ml of H₂SO₄ was added. The blue color at the interface showed the presence of carotenoids. For the presence of flavonoids, 2 ml of the mucilage solution was mixed with 1 ml of the concentrated HCl and magnesium ribbon gave the pink tomato-red color. For alkaloids, an amount of 0.05 g of the mucilage in 2 ml of 1.5%v/v HCl was boiled on a water bath and six drops of the Dragendorff’s reagent were added. The orange precipitate indicated the presence of alkaloids.

Determination of antioxidative activity

DPPH radical scavenging activity

Free radical scavenging activity of the mucilage was determined by a modified DPPH assay (Manosroi et al., 2013). Briefly, 50 µl of five serial concentrations (0.001–10 mg/ml) of the mucilage and 50 µl of ethanolic solution of DPPH were added into each well of a 96-well microplate (Nalge Nunc International, Penfield, NY). The mixtures were allowed to stand for 30 min at 25 ± 2 °C, and the absorbance was measured at 515 nm by a well reader (Bio-Rad, Model 680 Microplate Reader, Hercules, CA). Vitamin C (0.001–10 mg/ml) was used as a positive control. The experiments were done in triplicate. The percentages of radical scavenging activity were calculated according to the following equation: where A is the absorbance of the control and B is the absorbance of the sample. The sample concentration providing 50% of scavenging (SC₅₀) was calculated from the graph plotted between the percentages of scavenging and the sample concentrations.

Metal ion-chelating activity

The metal ion-chelating activity of the mucilage was assayed by the modified ferrous ion-chelating method (Manosroi et al., 2013). Briefly, 100 µl of five serial concentrations of the mucilage at 0.001–10 mg/ml were added to 50 µl of 2 mM FeCl₂ solution. The reaction was initiated by the addition of 50 µl of 5 mM ferrozine and the total volume was adjusted to 300 µl by distilled water. The mixture was left at 25 ± 2 °C for 15 min. Absorbance of the resulting solution was then measured at 570 nm by a microplate reader. EDTA (0.001–10 mg/ml) was used as a positive control. The negative control contained FeCl₂ and ferrozine. All experiments were performed in triplicate. The inhibition percentages of ferrozine-Fe2⁺ complex formation were calculated by the following equation: where A is the absorbance of the control and B is the absorbance of the sample. The sample concentration providing 50% metal ion-chelating activity (MC₅₀) was calculated from the graph plotted between the percentages of metal chelating activity and the sample concentrations.

Cytotoxicity on normal human gingival fibroblasts

Cell culture

Normal human gingival fibroblasts were obtained from the gingival tissue by explants techniques at Faculty of Dentistry, Chiang Mai University in Chiang Mai, Thailand. They were cultured in 30 mm diameter tissue culture dishes in the complete culture medium containing α-Modified Eagles culture medium (MEM-Alpha, Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone, UT), penicillin (100 U/ml; MEM-Alpha, Hyclone, UT), and streptomycin (100 mg/ml; MEM-Alpha, Hyclone, UT). Cells were incubated in a temperature-controlled and humidified incubator (Shel Lab, model 2123TC, Cornelius, OR) with 5% CO₂ at 37 °C and subcultured every 5–7 d. The cells at 5th–8th passage were used.

Cytotoxicity by SRB assay

The standards vitamin C was diluted in α-Modified Eagles culture medium to the final concentrations of 0.0001–1 mg/ml. The mucilage was reconstituted in distilled water to obtain the viscosity of 9 cP and assayed for cell proliferation activity on normal human gingival fibroblasts by SRB assay. The cells were plated at the density of 1.0 × 10⁵ cells/ml in 96-well plates and left for cell attachment overnight at 37 °C in 5% CO₂ incubator. Cells were then incubated with the mucilage (at the concentration that gave 9 cP viscosity) for 24 h. After incubation, the adherent cells were fixed in situ with 50% trichloroacetic acid, washed and dyed with 0.4% SRB. The bound dye was solubilized by Tris solution and the absorbance was measured at 540 nm in a microplate reader (Bio-Rad, Model 680 Microplate Reader, Hercules, CA). The results were presented as the percentages of cell viability which were calculated as follows: where T_treat indicated the OD₅₄₀ value of the treated cells after 24 h treatment and T_control indicated the OD₅₄₀ value of the control.

Determination of wetting time

Wetting times measurement of the mucilage were modified from previous study (Patil & Raghavendra Rao, 2011). Briefly, a filter paper (Whatman® No. 1) was placed in a Petri dish containing 6 ml of distilled water, human saliva or mucilage at 9 cP viscosity. A tablet with methylene blue dye powder on the upper surface was carefully placed on the filter paper in the Petri dish. The time required for water, saliva, or mucilage to reach the upper surface of the tablet and colored by wetting with the methylene blue solution completely was noted as the wetting time.

Determination of anti-adherent activity of Streptococcus mutans

Bacterial culture

Streptococcus mutans was inoculated into the tryptic soy broth and incubated at 37 °C, 5% CO₂ for 24 h. The absorbance at 550 nm (OD₅₅₀) was measured and the concentration of the culture was adjusted to obtain the OD₅₅₀ value of 0.5. The bacterial concentration was diluted one-fold to the bacterial concentration of 1 × 10⁶ CFU/ml.

Sample preparation

The top 10 mucilages which showed pseudoplastic non-Newtonian flow, high % yield, slightly or no cytotoxicity, and short wetting time were tested for anti-adherent activity. The reconstituted mucilages (at the viscosity of 9 cP) and human saliva were used. Briefly, the mucilage was reconstituted in distilled water and homogenized for 15 min. For human saliva, 20 ml of saliva was collected from human volunteers, centrifuged at 10 000g, 4 °C for 15 min and collected the clear solution. Distilled water was used as the negative control. The mucilages, human saliva, and distilled water were filtered through a membrane filter (0.2 µm) before use.

Anti-adherent activity

Hydroxyapatite (HA) was dispersed in phosphate buffer (pH 6.8) at the concentration of 5 mg/ml. An amount of 200 µl of HA suspension was added into each well of a 96-well plate, centrifuged at 1000 g for 15 min and the supernatant was discarded. The human saliva (200 µl) was added into each well, incubated at 37 °C with shaking at 80 rpm for 120 min and centrifuged at 1000g for 15 min. HA was washed by phosphate buffer solution. The 100 µl of S. mutans were mixed with 100 µl of mucilage or human saliva or distilled water. The mixture was incubated at 37 °C with shaking at 80 rpm for 120 min and then incubated without shaking at 37 °C for 60 min. The plate was centrifuged at 1000 g for 15 min and washed with phosphate buffer. The resazurin solution (100 µl) was added into each well and incubated at 37 °C with shaking at 80 rpm for 60 min. The fluorescence intensity was measured by a spectrofluorometer (Jusco, Hachioji, Japan) with the excitation/emission of 562/595. The % adhesion was calculated in comparing to the control (distilled water) as follows: where F_control is the fluorescence intensity of the control and F_sample is the fluorescence intensity of the sample.

Results and discussion

Screening of plant mucilage

Only 12 mucilages from five plants out of 23 plants could be reconstituted in distilled water at the concentration of 1% (w/v) to determine the viscosity and rheology (Table 1). Seven mucilages from O. citriodorum (25 and 60 °C), A. heterophyllus (25 and 60 °C), A. esculentus (25 and 60 °C), and B. alba (25 °C) demonstrated pseudoplastic non-Newtonian flow similar to the normal human saliva. A. esculentus (60 °C) mucilage showed the lowest concentration for the 9 cP viscosity, with SVEU of 2.00–2.50 in comparing with 0.2% SCMC (1 SVEU). The phytochemical screening demonstrated the presence of glycosides and alkaloids in almost all the selected seven mucilages. The detected alkaloid might be from the presence of glycoprotein in the plant mucilage. This may be due to the chemical structure of alkaloid composing nitrogen atom-linked pyridine and/or pyrrolidine ring, which are similar to some amino acids such as proline and tryptophan in protein molecules (García-Mateos et al., 1996). Anthraquinone, carotenoid, and tannin were not detected in all mucilages. The mucilages from O. citriodorum, A. heterophyllus, A. esculentus, and B. alba produced the pseudoplastic non-Newtonian flow and were further developed for the mucilage extraction processes.

Table 1. Percentage yields, viscosity, and rheology of the mucilages from 23 Thai plants extracted by water at two different temperatures.

Plants	Extraction method	Percentage yields (%, dry weight)	Concentration at 9 cP (%w/v)	Saliva viscosity equivalent unit (SVEU)	Rheology
Abelmoschus esculentus Linn.	Water, 25 °C	0.785	0.10–0.13	1.54–2.00	NNPs
	Water, 60 °C	0.789	0.08–0.10	2.00–2.50	NNPs
Artocarpus heterophyllus Lam.	Water, 25 °C	0.452	0.30–0.35	0.57–0.67	NNPs
	Water, 60 °C	0.440	0.15–0.20	1.00–1.33	NNPs
	Basella alba Linn. (flower)	Water, 25 °C	0.723	0.25–0.50	0.40–0.80	NNPs
Water, 60 °C		0.440	0.50–1.00	0.20–0.40	NNP
Basella alba Linn. (treetop)		Water, 25 °C	1.780	>1.00	ND	NNP
	Water, 60 °C	2.562	0.75–1.00	0.20–0.27	NNP
Musa sapientum Linn.	Water, 25 °C	0.741	0.50–0.70	0.28–0.40	NNP
	Water, 60 °C	2.898	0.75–1.00	0.2–0.27	NNP
	Ocimum citriodorum Vis.	Water, 25 °C	0.020	0.40–0.46	0.43–0.50	NNPs
Water, 60 °C		0.009	>1.00	ND	NNPs
Sodium carboxymethylcellulose		–	–	0.20	1.00	NNPs

ND, not detected; NNP, non-Newtonian, plastic; NNPs, non-Newtonian, pseudoplastic.

Viscosity, rheology, and phytochemical determination

Viscosity and rheology of 40 mucilages of four plants (O. citriodorum, A. heterophyllus, A. esculentus and B. alba) using 10 extraction methods showed that 27 mucilages gave the pseudoplastic non-Newtonian flow similar to human saliva (Table 2). The lowest mucilage concentration of 0.08–0.10% (w/v) at 9 cP was observed in the A. esculentus (60 °C) mucilage with the SVEU of 2.00–2.50, which was lower than the concentration of mucin (0.5% w/v) in the commercial artificial saliva of about five times (Hahnel et al., 2009). The different in rheological behaviors might be due to the different hydrolyses of the intermolecular bonds of sugars in the mucopolysaccharide of the mucilage by the different temperatures or pHs used in the extraction procedures (Trofimova & Babkin, 2010). In fact, the major composition responsible for the viscosity of the mucilage is pectin, a polysaccharide which consists mainly d-galacturonic acid units, joined in chains by means of α-(1-4) glycosidic linkage (Sriamornsak, 2003). However, pectin with high concentration (higher 4% w/w) has been previously reported to have the non-Newtonian behavior, whereas low concentration gave the Newtonian flow (Charcosset & Choplin, 1996). Thus, the pseudoplastic non-Newtonian flow of the plant mucilage in this study might be due to the mucin, a mucopolysaccharide containing N-acetylglucosamine and sugar chain which is usually found in many plants. Chemical structure of mucin composes of polysaccharide and amino acid with a nitrogen atom. The plant mucilage which gave positive results with alkaloid was thus resulted from the nitrogen atom containing chemical constituents.

Table 2. Percentage yields, viscosity and rheology of 40 mucilages from the four selected Thai plants by various extraction processes.

Plants	Extraction method	Percentage yields (%, dry weight)	Concentration at 9 cP (%w/v)	Saliva viscosity equivalent unit (SVEU)	Rheology
Abelmoschus esculentus Linn.	Water, 25 °C	0.79	0.10–0.13	1.54–2.00	NNPs
	Water, 60 °C	0.79	0.08–0.10	2.00–2.50	NNPs
	Water, 100 °C	1.12	0.80–1.0	0.20–0.25	NNPs
	Water, pH 2	0.46	0.05–0.10	2.00–4.00	NNPs
	Water, pH 11	0.51	0.15–0.20	1.00–1.33	NNPs
	Microwave, 1 min	0.63	0.10–0.20	1.00–2.00	NNPs
	Microwave, 3 min	0.77	0.10–0.20	1.00–2.00	NNPs
	Microwave, 5 min	0.57	0.20–0.30	1.67–1.00	NNPs
	Steam bath	2.79	>1.00	ND	NNPs
	Tris-HCl buffer	1.61	0.20–0.25	0.80–1.00	NNPs
Artocarpus heterophyllus Lam.	Water, 25 °C	0.45	0.30–0.35	0.57–0.67	NNP
	Water, 60 °C	0.48	0.15–0.20	1.00–1.33	NNPs
	Water, 100 °C	2.76	–	–	–
	Water, pH 2	0.15	0.50–0.75	0.27–0.40	NNPs
	Water, pH 11	0.46	0.06–0.13	1.54–3.33	NNP
	Microwave, 1 min	1.41	–	–	–
	Microwave, 3 min	0.97	>1.00	ND	NNPs
	Microwave, 5 min	0.95	>1.00	ND	NNPs
	Steam bath	9.64	–	–	–
	Tris-HCl buffer	0.59	0.10–0.20	1.00–2.00	NNPs
Basella alba Linn.	Water, 25 °C	0.72	0.25–0.50	0.40–0.80	NNPs
	Water, 60 °C	0.44	0.50–1.00	0.20–0.40	NNP
	Water, 100°C	0.79	0.50–0.75	0.27–0.40	NNP
	Water, pH 2	0.25	0.45–0.50	0.40–0.44	NNPs
	Water, pH 11	0.32	0.40–0.50	0.40–0.50	NNPs
	Microwave, 1 min	0.49	0.40–0.50	0.40–0.50	NNPs
	Microwave, 3 min	0.23	0.50–0.60	0.33–0.40	NNPs
	Microwave, 5 min	0.44	0.50–0.60	0.33–0.40	NNPs
	Steam bath	3.32	–	–	–
	Tris-HCl buffer	0.75	0.65–0.75	0.27–0.31	NNPs
Ocimum citriodorum Vis.	Water, 25 °C	0.12	0.40–0.46	0.43–0.50	NNPs
	Water, 60 °C	0.07	>1.00	ND	NNPs
	Water, 100 °C	0.09	–	–	–
	Water, pH 2	0.21	>1.00	ND	NNP
	Water, pH 11	0.12	1.00	0.2	NNPs
	Microwave, 1 min	0.79	0.70–0.80	0.25–0.28	NNP
	Microwave, 3 min	0.83	1.00	0.2	NNP
	Microwave, 5 min	0.46	0.70–0.80	0.25–0.28	NNP
	Steam bath	2.22	0.40–0.50	0.40–0.50	NNPs
	Tris-HCl buffer	0.64	0.30–0.50	0.40–0.67	NNPs
Sodium carboxymethyl cellulose		–	0.20	1.00	NNPs

ND, the viscosity was not detected at the concentration of 1% w/v; NNP, non-Newtonian, plastic; NNPs, non-Newtonian, pseudoplastic.

Antioxidative activities and cytotoxicity

The antioxidative activities was carried out to evaluate the capacity to eliminate free radicals from the dental surfaces. Antioxidative activities of 27 mucilages which gave the pseudoplastic non-Newtonian flow are shown in Table 3. The mucilages from O. citriodorum, A. esculentus, and B. alba exhibited antioxidative activities including DPPH radical-scavenging and metal-chelating activities. The highest DPPH radical-scavenging and metal-chelating activities were observed in the mucilages from B. alba (microwave, 3 min) and A. esculentus (microwave, 1 min) with the SC₅₀ and MC₅₀ values of 0.71 ± 0.32 and 1.11 ± 0.52 mg/ml (1.01- and 16.86-folds of vitamin C and EDTA), respectively. This might be due to the presence of alkaloid and glycoside which have been previously demonstrated to have significant antioxidative activities (Jagan Mohan Rao et al., 2007). In addition, in vitro antioxidative activities of the plant mucilages such as the DPPH radical-scavenging (Chatchawal et al., 2010; Lin et al., 2005; Nagai et al., 2006) and metal ion-chelating activities (Corgano et al., 2011; Wang et al., 2011) have been reported. The cytotoxic effect on normal human gingival fibroblasts of the 27 plant mucilages represented by the percentages of cell viability is shown in Table 3. Most mucilages exhibited slightly or no cytotoxicity on normal human gingival fibroblasts except those from B. alba (microwave, 3 and 5 min), A. esculentus (100 °C), O. citriodorum (25 °C), and A. esculentus (60 °C) which demonstrated high cytotoxicity with cell viability of less than 30%. Thus, different conditions including temperature and pH used in the extraction process might affect the chemical constituents containing in the mucilages thereby resulting different biological activities and cytotoxicities.

Table 3. Antioxidative activities and cytotoxicity of the 27 mucilages by different extraction processes from the four selected Thai plants shown the pseudoplastic non-Newtonian flow rheology.

		Antioxidative activities
Plants	Extraction method	SC50	MC50	Cell viability (%)
Abelmoschus esculentus Linn	Water, 25 °C	2.85 ± 0.23	ND	176.38 ± 19.86
	Water, 60 °C	2.66 ± 0.21	5.39 ± 4.99	168.57 ± 24.76
	Water, 100 °C	5.91 ± 0.45	10.86 ± 2.46	15.71 ± 0.86
	Water, pH 2	3.72 ± 0.64	ND	115.43 ± 46.76
	Water, pH 11	ND	7.25 ± 3.02	108.28 ± 4.53
	Microwave, 1 min	ND	1.11 ± 0.52	79.62 ± 1.19
	Microwave, 3 min	ND	ND	84.67 ± 17.45
	Microwave, 5 min	ND	ND	89.14 ± 8.98
	Steam bath	3.27 ± 0.14	ND	112.09 ± 5.74
	Tris-HCl buffer	6.46 ± 1.95	ND	121.52 ± 24.89
Artocarpus heterophyllus Lam.	Water, 60 °C	ND	ND	18.19 ± 4.06
	Water, pH 2	ND	ND	99.71 ± 69.31
	Microwave, 3 min	ND	ND	147.43 ± 31.22
	Microwave, 5 min	ND	ND	138.57 ± 16.68
	Tris-HCl buffer	ND	ND	172.95 ± 15.31
Basella alba Linn.	Water, 25 °C	4.85 ± 1.66	ND	105.81 ± 72.32
	Water, pH 2	ND	ND	73.43 ± 31.71
	Water, pH 11	ND	1.68 ± 0.70	113.52 ± 8.03
	Microwave, 1 min	4.67 ± 1.07	2.33 ± 0.88	134.76 ± 17.07
	Microwave, 3 min	0.71 ± 0.32	4.69 ± 0.89	18.19 ± 1.84
	Microwave, 5 min	13.84 ± 0.14	ND	23.90 ± 10.43
	Tris-HCl buffer	20.63 ± 5.53	ND	63.52 ± 24.56
Ocimum citriodorum Vis.	Water, 25 °C	ND	1.53 ± 0.72	14.28 ± 2.98
	Water, 60 °C	ND	ND	110.76 ± 39.36
	Water, pH 11	ND	ND	91.53 ± 24.89
	Steam bath	2.66 ± 0.41	4.88 ± 2.94	88.19 ± 10.14
	Tris-HCl buffer	ND	ND	100.67 ± 11.31
Standard vitamin C		0.72 ± 0.00	NA	NA
Standard EDTA		NA	18.71 ± 16.54	NA

NA, not applicable; ND, not detected; Cell viability > 90% = non-toxic, 60–90% = slightly toxic, 30–59% = moderately toxic, and <30% = highly toxic.

Wetting time and anti-adherent activity

The wetting times of 27 mucilages compared with distilled water and normal human saliva were investigated. Normal human saliva demonstrated the wetting time of 10.87 ± 1.79 min. Twenty-five mucilages exhibited shorter wetting time than that of the human saliva with the shortest wetting time of 2.75 ± 0.51 min observed in the mucilage of A. esculentus (microwave, 5 min). The wetting time is one of the important properties of the mucilage that is needed for artificial saliva preparation. Loss or reduction in saliva production of the xerostomia patients will result in the difficulty of food swallowing and speaking, the increasing in the development of plaque and dental caries, and also oral candidiasis. These problems can be improved by the application of the artificial saliva which has short wetting time that foods can be wet in a few seconds or minutes to make it easier to chew and swallow (Lagdive et al., 2011).

Streptococcus mutans can colonize the tooth surface and initiate plaque formation. The mucilages which inhibit the adsorption of S. mutans-binding to HA surfaces can lead to the prevention of dental caries and periodontal diseases arising out of dental plaque formation. The top 10 mucilages including mucilages from O. citriodorum (steam), A. heterophyllus (Tris-HCl buffer and microwave, 3 min), A. esculentus (25 and 60 °C, pH 2 and 11, steam and Tris-HCl buffer), and B. alba (microwave, 5 min) were investigated for anti-adherent activity. Normal human saliva demonstrated an increase in adhesion of S. mutans on HA beads of 36.74 ± 5.85%. The mucilages from O. citriodorum (steam), A. heterophyllus (microwave, 3 min), and A. esculentus (pH 11) exhibited the decrease in adhesion of S. mutans on HA beads of 4.88 ± 5.96, 3.97 ± 4.74, and 5.39 ± 9.70%, respectively, whereas the remaining seven mucilages showed slightly increased the adhesion, but lower than the normal human saliva as shown in Table 4. This might be due to the amino sugars such as glucosamine in the plant mucilage (Metaxatos et al., 2003) which can inhibit the binding of S. mutans to the saliva-coated HA (Hajishengallis et al., 1992). Usually, the alkaline extraction gave higher yield than the acid extraction because of the more hydrolysis occurring in the acid extraction. Acid hydrolysis can cause some loss of the polysaccharide containing in the mucilage. However, the previous study has demonstrated that the alkaline extraction can result in the destruction of cell walls and breakdown of the coarse-fiber structure, which becomes looser and more release of the polysaccharides (Huang et al., 2010). Thus, mucilages from alkaline extraction are composed of various compounds such as polysaccharides, celluloses, hemi-celluloses, and lignin which may have synergistic antiadherent activity of S. mutans on HA beads. The results from this study demonstrated the potential application for artificial saliva preparation from mucilages of some Thai plants which have similar viscosity and rheology to normal human saliva with high antioxidant activity, no cytotoxicity, short wetting time, and having S. mutans adhesion inhibition on HA.

Table 4. The top 10 mucilages from the four selected Thai plants with the highest anti-adherent activity of S. mutans on HA beads.

Plants	Extraction method	% Decrease	% Increase
Abelmoschus esculentus Linn.	Water, 25 °C	–	1.91 ± 2.25
	Water, 60 °C	–	2.02 ± 1.67
	Water, pH 2	–	36.63 ± 7.28
	Water, pH 11	5.39 ± 9.70	–
	Steam bath	–	4.71 ± 6.15
	Tris-HCl buffer	–	23.63 ± 28.90
Artocarpus heterophyllus Lam.	Microwave, 3 min	3.97 ± 4.74	–
	Tris-HCl buffer	–	4.57 ± 4.60
Basella alba Linn.	Microwave, 5 min	–	4.83 ± 3.47
Ocimum citriodorum Vis.	Steam bath	4.88 ± 5.96	–
Normal human saliva		–	36.74 ± 5.85

The optimization of the mucilage extraction was the mucilage from Ocimum citriodorum extracted by bath steaming since the obtained mucilage exhibited pseudoplastic non-Newtonian behavior, antioxidative activities (including DPPH radical scavenging and metal chelating activities), and also anti-adherent activity of S. mutans on HA beads.

Conclusion

Twenty-seven mucilages extracted from four out of 23 plants exhibited pseudoplastic non-Newtonian flow, which was similar to human saliva. Phytochemical constituents including glycoside and alkaloid were found in most mucilages. The mucilages from B. alba (microwave, 3 min) and A. esculentus Linn. (microwave, 1 min) gave the highest DPPH radical-scavenging and metal-chelating activities of 1.01- and 16.86-folds of vitamin C and EDTA, respectively. Most mucilages demonstrated no cytotoxic effect on normal human gingival fibroblasts with shorter wetting time than normal human saliva of 1.02- to 3.95-folds. The mucilages from O. citriodorum (steam), A. heterophyllus (microwave, 3 min), and A. esculentus (pH 11) could inhibit the adhesion of S. mutans on HA beads of 4.88 ± 5.96, 3.97 ± 4.74, and 5.39 ± 9.70%, respectively, whereas the normal human saliva increased the adhesion of 36.74 ± 5.85%. The steam extraction is the best extraction process to prepare the mucilages because it is the simple process. The mucilage from this process was concentrated since there was no water dilution. Also, the highest yields of the mucilages from all plants were obtained from this process. In addition, the mucilage from Ocimum citriodorum exhibited the pseudoplastic non-Newtonian behaviors, antioxidative activities (including DPPH radical-scavenging and metal-chelating activities) and also anti-adherent activity of S. mutans on HA beads. These results indicated the potential application of mucilages from these plants for the preparation of artificial saliva.

Declaration of interest

The authors report that they have no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Thailand Research Fund (TRF), Manose Health and Beauty Research Center, the Natural Product Research and Development Center (NPRDC), and Science and Technology Research Institute (STRI), Chiang Mai University (Chiang Mai, Thailand).