Reserch and Patent

Introduction

The nest of Swiftlet (Collocaliini) has been empirically known for its beauty effect and infectious disease prevention effects since ancient times and is also used as a premium ingredient in China. It is well known that the nest of Swiftlet contains high levels of sialic acid. Recent studies have shown that sialic acid induces the production of IGF-1 and thus participates in maintaining the homeostasis of systemic function. Saber et al. 1) confirmed a close association between serum IGF-1 and the risk of cerebral infarction and myocardial infarction through the Framingham study. It is generally believed that in order to prevent the onset of cerebral infarction and myocardial infarction in advance, it is necessary to control the decline of serum IGF-1 in the middle-aged and elderly range.
During the exploration of sialic acid peptides with antiviral activity, Matsumoto et al. 2) found there was no correlation between sialic acid content and antiviral activity, but the sugar chain structure centered on the binding site of sialic acid was effective. Golden pill 3) also reported that sialic acid binding site is very important.
Therefore, we believe that the structure of functional participation components from sialic acid derived from swiftlet nests is also important, and the functionality of the obtained sialic acid in the study of manufacturing methods are examined.

1. Extract Sialic Acid from Swiftlet Nests

The sugars of sialic acid in animals were modified with Asn type, mucin type and proteoglycan type, and the binding mode of amino acid residues was β-binding. The sialic acid in swiftlet nest is mucin type and exists in the non-reducing end of sugar chain bound with Asn residue. The sugar chain is a β-bond, and the oligosaccharides containing sialic acid (sialic acid oligosaccharide: SAO®) can be separated from the glycosaminoglycan by β-dissociation.
In order to obtain SAO® from swiftlet nest, it is necessary to extract it under alkaline and acidic conditions. In other words, 10 times of 0.25% (w/w) sodium hydroxide aqueous solution was added to the swiftlet nest to be extracted under alkaline conditions. After cooling, the insoluble substance was removed by centrifugation and filtration. Then it was desalted by electrodialysis to obtain dry powder.The yield was about 30%. In addition, 10 times of 0.5M hydrochloric acid solution was added to swiftlet nest to be extracted under acid condition. The yield was about 8%. The extraction of SAO® was confirmed by gel filtration. Please see Figure 1. Figure 1(1) shows the value of sialic acid and sugar extracted under acidic conditions, and (2) shows the value of sialic acid and sugar extracted under alkaline conditions. In acid extraction, the peak value of sialic acid was not correlated with the peak value of sugar, and the peak values were consistent under alkaline extraction. It can be seen that the SAO® can be extracted from swiftlet nest under alkaline condition. 

 

Figure 1: Status of Sialic Acid Oligosaccharides Extracted from swiftlet nest.

Column: Toyopearl HW 40 S (10 x 800mm)
Eluate: 50% ethanol solution
Flow Velocity: 1 ml/ml
Sorting: 200 drops/bottle

2. Functionality of SAO® Derived from Swiftlet Nest

Swiftlet nest has been eaten since ancient times as a nourishing tonic ingredient, but it has not been clarified as a functional nutrient until recent years. It was widely believed that sialic acid in swiftlet nest played a role in the 1950s. However, although its functional expression mechanism was unknown, sialic acid has recently begun to attract attention again from anti-aging studies. In 2009, Malicdan 4), Noguchi 5), Gangdao 6) respectively clearly proposed that sialic acid can induce IGF-1 production, and found a variety of physiological and pharmacological functions. Therefore, it is considered that the functional expression of swiftlet nest is not directly induced by cell proliferation factors, but produced by some kind of signal in vivo.
Therefor the effect of SAO® on human normal cell proliferation was measured and shown in Table 1. The cell proliferation rate of SAO was calculated when the addition amount of SAO® was 0 mg/ml (blank) as 100%. The proliferation capacity of the cells, usually found in sialic acid, was observed in a concentration-dependent manner by SAO®. However, since this phenomenon is also observed in sialic acid standard, it is not believed that this is due to the effect of swiftlet nest. As is shown in Figure 2, because it has been clarified that sialic acid produces IGF-1, and the function of SAO® is determined by measuring IGF-1. In addition, the ability to produce various growth factors other than IGF-1 was SAO® measured, as shown in Table 2. In addition to IGF-1, the production of FGF-β (Table 2-1), EGF (Table 2-2) and HGF (Table 2-3) were increased excluding PDGF (Table 2-4). The rate of increase is over 50%. Since PDGF is a proliferation factor derived from platelets, the effect of PDGF on the production of PDGF can not be confirmed in the culture system, but different results can be obtained in the biological system (in vivo). The cell activity of SAO® was significantly higher than that of standard free sialic acid (FAS) used as standard product.

Table 1: Proliferation Rate of SAO on Human Body Fibroblasts

When the addition of SAO® was 0 mg/ml (blank), the cell proliferation rate of each concentration.

Figure 2: Effects of SAO® on IGF-1 Production in Normal Human Cells

Table 2: Effects of SAO® on Productions of Various Growth Factors in Normal Human Cells

Table 2-1: FGF-b

Table 2-2: EGF

Table 2-3: HGF

Table 2-4: PDGF

Organisms accumulate cells with various functions. The gaps between cellular cells is filled with Extra cellular matorix (ECM), which maintains normal ECM and inhibits the development of various chronic refractory inflammatory diseases. However, the proteins, polysaccharides and lipids that make up the ECM are prone to be denatured and are vulnerable due to psychological, physical, environmental and age-related factors. In general, the mechanism from destruction to regeneration is a very important process in the normalization of tissue, but if the regeneration process is impaired, it will slowly develop into chronic disease and significantly reduce the quality of life (QOL). Typical chronic inflammatory diseases include rheumatoid arthritis, ulcerative enteritis, and Crohn's disease and other inflammatory bowel diseases and multiple sclerosis. These diseases are mainly treated with immunosuppressants, but long-term use can cause more serious side effects such as infection and cancer.
For these reasons, it is important to suppress ECM damage and obstacles during regeneration. It is almost impossible to analyze the effect of SAO on the total components of ECM, so the effect of SAO® on collagen and glucomannan (GAG) containing hyaluronic acid are discussed.
Using normal fibroblast-forming cells of human body, the effect of SAO® on the main protein type-1 collagen of ECM was measured, as shown in Figure 3. In addition to 5 pg/ml standard sialic acid, the production of type 1 collagen was increased by SAO® 10 times compared with no addition, and the result of adding 5 pg/ml as sialic acid was 0.532, which increased more than 20 times compared with 0.0251 without addition. The results showed that the SAO® addition amount of 1 mg/ml, which could promote the proliferation of cells by 10%, promotes the production of heparin-binding proliferation factor (PDGF-1, FGF-β, EGF, HGF), and the production of collagen and hyaluronic acid (GAG).

Figure 3: Effect of SAO® on Type 1 Collagen

Conclusion

Swiftlet nest (Collocaliini) has been known for its beauty benefits and the prevention of infectious diseases since ancient times. This year, its functional component- sialic acid has also attracted a lot of attention. It is well known that sialic acid induces the production of IGF-1, which is closely related to the maintenance and improvement of systemic function. However, the content of sialic acid has no obvious relationship with antiviral activity, and the sugar chain structure centered on sialic acid binding site is very important. As a result, the structure of functional components from sialic acid in swiftlet nest is of great importance, so the method of extracting was examined, and it was found that SAO® can be extracted under alkaline condition. The molecular weight of the sialic acid oligosaccharide is 1.7k~2.3kDa, and the highest activity can be observed when the massive ratio of sialic acid/hexose is between 0.5~0.9.
So-called heparin binding growth factors such as IGF-1, FGF-β, EGF and HGF can be induced by SAO®. In addition, although no data were shown, it was also observed to induce the production of total collagen.
These results suggest that SAO® from swiftlet nest is a very important material to maintain the stability of the organism by producing various growth factors and preventing the destruction of the extracellular basement membrane 7). Especially with the growth of age, IGF-1 also decreases in people in their 60s, which is about 1/2 of those in their 20s. It can be inferred that this phenomenon may be the cause of the so-called aging disease, on this basis, we believe that SAO® derived from swiftlet nest is the first choice to participate in the control of IGF-1 reduction.

Reference

1）Saber H., et. al.: Stroke, 48(7), 1760-1765 (2017)
2) Matsumoto Guangqing et al.: Journal of the Japanese Society of Animal Production, 73(1), 49-56(2002)
3）Kanamaru Y., et. al.: Biosci. Biotechnol. Biochem., 63 (1), 246-249 (1999)
4）Malicdan M. C., et. al.: Nature Medicine, 15(6), 690-695 (2009)
5) Noguchi: Biochemistry, 83(4), 316-320(2011)
6) Okadawa Takashi: Fragrance J., 37(10), 43-47(2009)
7) Gu Jiudian et al.: Pharmacology and Clinical Study, 27(4), 187-193(2017)