After water imbibition, the outer layer seed coat of shaddock ( Citrus grandis Osbeck) produces transparent gel-like mucilage (MSS), but its characteristics have never been studied before. This study aimed to assess the physico-chemical and functional properties of MSS. Extractions of MSS with deionized water at room temperature yielded about 3.5% based on the dry weight of seed. The major components were neutral sugars and uronic acids in the amounts of 33.5% and 49.6%, respectively. The acidic nature of MSS was confirmed by ruthenium red staining. Its water holding capacity and viscosity were 44.53 g•g -1 DW and 1660 cP at 10 g/L, respectively. MSS showed a weak quenching activity against DPPH radical, and moderate ferrous ion-chelating and superoxide anion radical scavenging activities, with IC 50 value of 1.5 g/L and 1.1 g/L, respectively. A methyl thiazolyl tetrazolium (MTT) assay demonstrated that MSS significantly stimulated the viability of mouse skin fibroblasts (NIH/3T3) at 5 - 300 mg/L. These results impart the potential usefulness of the MSS to food, cosmetics and other applications.
Many angiosperms, including Brassicaceae, Solanaceae, Linaceae, and Plantaginaceae, among others, produce a pectinaceous mucilage layer in their outer seed coat, known as myxospermy [
Mucilage is a complex heterogeneous polysaccharide. Polysaccharides have emerged as an important class of bioactive natural products [
Shaddock (Citrus grandis Osbeck) is an important economic fruit in Taiwan during autumn. Our preliminary study indicated that the outer seed coat of shaddock contained a high amount of transparent gel-like mucilage (MSS) and had never been studied before. In general, the seeds are removed and discarded while the pulp is eaten. The objective of this paper was to characterize MSS with respect to its physicochemical properties such as the viscosity, water holding capacity, antioxidative potency in vitro and cytotoxicity against the mouse embryo fibroblast (NIH 3T3) cells. Such information contributes toward the sustainable reuse of agricultural wastes and imparts the potential usefulness of MSS to various applications.
The seeds were removed from ripe shaddock fruits (Citrus grandis Osbeck) that were purchased from a local market in the northern Taiwan. After being dried at room temperature, they were stored in a desiccator box until use.
The dried seeds were imbibed in water for about 15 min to form a gel around wetted seed. Then, they were incubated with the cationic dye ruthenium (2 g/L).
The dried seeds were extracted three times with water (water to seed ratio of 5:1) on a reciprocal shaker (BT-150, Yihdern, Taiwan) under the room temperature for 20 min each time. The extracts were mixed and vacuum filtrated through a glass microfibre filter (Whatman GF/A, GE Healthcare, USA). The filtrate was lyophilized in a freeze dryer (DM-25ES, VirTis, NY, USA). The dry MSS yield was estimated.
Ash content was determined using the AOAC oven method [
The viscosities of MSS solutions (1 - 10 g/L) were measured using Dial Reading viscometer (LVT, Brookfield, Massachusetts, USA) with spindle No. 18 at 26˚C. The viscosities of xanthan (1 - 5 g/L) and gum arabic (1 - 10 g/L) were compared.
The WHC of MSS was determined by an adaptation of the filtration method of Robertson and Eastwood [
The antioxidant activity of the MSS was evaluated by using the DPPH free radical scavenging method described by Yamaguchi et al. [
Briefly, 20 mL of the MSS or butylated hydroxytoluene (BHT) at various concentrations were mixed with 100 mM Tris-HCl buffer (pH 7.4, 80 mL), and added to 100 mL DPPH radical in ethanol (0.25 mM). The mixture was shaken vigorously and left to stand for 20 min at room temperature in the dark. The absorbance was measured at 517 nm. The percentage of DPPH radical scavenging activity was calculated as [(A0 − A1)]/A0 ´ 100, in which A0 is the absorbance of the control, and A1 is the absorbance of the MSS and BHT.
For chelating effect determination, briefly, 100 mL of the MSS or EDTA at various concentrations were mixed with 2 mM ammonium ferrous sulphate (10 mL). The reaction was initiated by the addition of 5 mM ferrozine (20 mL) and the mixture was shaken vigorously and left standing at room temperature for 10 min, and the absorbance of the mixture was determined at 562 nm. The percentage of inhibition of ferrozine-ferrous complex formation was calculated by [(A0 − A1)]/A0 ´ 100, in which the notations of A0 is the absorbance of the control, and A1 is the absorbance of the MSS and EDTA.
For superoxide anion radical scavenging determination, 100 mL of the MSS or ascorbic acid at various concentrations were mixed with 100 mL NBT solution (300 mM), 100 mL PMS solution (120 mM), and 100 mL NADH solution (936 µM). The mixture was shaken vigorously and left to stand for 5 min at room temperature in the dark, and the absorbance of the mixture was determined at 560 nm. The percentage of superoxide anion radical scavenging activity was calculated as [(A0 − A1)/A0] ´ 100, in which A0 is the absorbance of the control, and A1 is the absorbance of the MSS and ascorbic acid.
Mouse embryonic fibroblasts (cell line NIH 3T3, BCRC 60071) were purchased from the Bioresource Collection and Research Centre in Hsinchu (Taiwan).
NIH-3T3 cells (1 ´ 104/well) in their exponential growth phase were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 5% CO2 at 37˚C. Following a 12 h incubation period, the cells were exposed to various concentrations (5 - 300 mg/L) of the MSS solution that was filtrated through a filter (PTF205030, BIOFIL, Guangzhou, China) for another 12 h. The viability of cells was assessed using the MTT assay as described elsewhere. Briefly, the medium was aspirated and MTT was added to cells at a concentration of 0.25 g/L. Cells were incubated at 37˚C for 3 h and the formazan product was solubilized with dimethylsulfoxide. The absorbance was detected in the microplate reader at 570 nm. Concanavalin A (ConA) (5 mg/mL) was the positive control.
The results are presented as means of at least three replicates ± standard error (SE). The data were subjected to one-way ANOVA and the differences between means were measured at the 5% probability level using Fisher’s protected least significant difference (LSD) test (CoHort Software, Monterey, CA, USA).
After imbibition at room temperature for 15 min, the transparent gel-like mucilage appeared at the surrounding of seed coat, and its thickness was approximately 30% of the seed width at the middle of its length (
A simple aqueous extraction process could obtain MSS. The yield of MSS was approximately 3.5% of the dry mass of the whole seed (
Parameter | Quantity |
---|---|
Yield* | 3.5% ± 0.4% |
Ash | 5.00% ± 0.06% |
Neutral sugar | 33.5% ± 0.6% |
Uronic acid | 49.6% ± 2.8% |
Protein | 0.7% ± 0.1% |
Total phenol | 0.2% ± 0.0% |
[
MSS was effective at increasing the viscosity of solutions at levels as low as 5 - 10 g/L, reached to 1660 cP at 10 g/L (
Similar to the trend of viscosity, the WHC analysis reveals that MSS could hold 44 times their weight of water (
These WHC and viscosity features are consistent with MSS ecological and physiological roles, such as facilitation of seed hydration [
The stable DPPH radical is a widely adopted target for evaluating the free radical scavenging ability of various samples [
Water holding capacity g of water/g DW | |
---|---|
Xanthan | 274.0 ± 45.8a |
MSS | 44.5 ± 3.7b |
Gum arabic | 7.9 ± 1.6b |
in the seeds of different grape species. Hence, a weak free radical scavenging ability may be attributed to MSS to contain low levels of phenol (
Iron is essential for oxygen transport, respiration and activity of many enzymes, which are required for sustaining life; however, ferrous, not ferric, is the most powerful pro-oxidant among the various species of metal ions and is able to generate free radicals from peroxides by the Fenton reaction to catalyze oxidative changes in lipids, proteins, and other cellular components [
Of the reactive oxygen species, superoxide anion radical is generated first [
MTT assay showed that MSS significantly stimulated the viability of mouse skin fibroblast NIH3T3 at 5 - 300 mg/L, reached to 130% increase at 100 mg/L, and the increasing efficiency was profoundly higher than ConA, which had only 20% increase, a value much lower than that of MSS (116%) at the same concentration (5 mg/L) (
The present work clearly demonstrated that MSS could be easily obtained via a simple extraction process from the outer layer seed of shaddock, and its yield was about 3.5% of the dry seed. MSS exhibited a moderate viscosity, water holding capacity, and ferrous ion-chelating as well as superoxide anion radical scavenging activities. MSS not only showed nontoxicity, but also stimulated the proliferation of NIH3T3 cells. These results impart the potential usefulness of MSS to food, cosmetics and other value-added applications. Several kinds of seed coat mucilage present in different plants, hence, another important subject will understand the composition and linkage of the purified MSS and further identify its polysaccharide type.
The authors would like to thank Professor Jong-Ching Su for helpful comments on drafting this manuscript and Associate Professor Jer-Chia Chang (Department of Horticulture, National Chung Hsing University) for identifying the species of shaddock. This work was supported by the grant NSC 101-2311-B-390-001 from the National Science Council, Executive Yuan, Taiwan.
Heng-Long Wang,Wei-Yu Chen,Pei-Jung Tsai,Chen-Yi Lin,Yuan-Ting Hsu,Li-Feng Chen,Wei-Zhi Wu,Wan-Chi Wang,Wen-Jen Yang,Chao-Lin Chang,Wei-Tung Liao, (2016) Isolation of Acidic Mucilage from the Outer Seed Coat of Shaddock (Citrus grandis Osbeck) and Evaluation of Its Functional Properties. American Journal of Plant Sciences,07,780-788. doi: 10.4236/ajps.2016.76072