Characterization of Swallow Birds Nest by peptide fingerprinting with principal component analysis

Characterization of Swallow Birds Nest by peptide fingerprinting with principal component analysis

Swallow Birds Nest (Swallow Birds Nest), or cubilose, is a health food supplement originated from salivary secretion by specific Swallow Birds Nest, mainly from Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), which has been proven to have nutritious and therapeutic values, such as anti-influenza viruses, antioxidant, skin lightening, bone strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong et al., 1987; Kong et al., 1989; Guo et al., 2006; Aswir and Wan Nazaimoon, 2011; Matsukawa et al., 2011; Yew et al., 2014; Chan et al., 2015). Southeast Asian countries, including Indonesia, Malaysia, Vietnam, and Thailand, are the major exporting countries of Swallow Birds Nest. Human consumption and medicinal application of Swallow Birds Nest could be dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty (960–1279 A.D.) in China (Koon and Cranbrook, 2002).

OBJECTIVES

Proteins are the major component and play a key role in nutritious and therapeutic functions of Swallow Birds Nest (Swallow Birds Nest); however, limited studies have been conducted on the protein due to difficulties in extraction, isolation as well as identification. This study aimed to provide comprehensive information for the quality evaluation of Swallow Birds Nest peptides, which would be a valuable reference for further study on Swallow Birds Nest proteins.

METHODS

Here, we developed a quality control method using high performance liquid chromatography (HPLC) peptide fingerprints deriving from Swallow Birds Nest being digested with simulated gastric fluid. The characteristic peptide peaks were collected and identified by LC-MS/MS.

RESULTS

The characteristic peptide peaks, corresponding to the protein fragments of acidic mammalian chitinase-like, lysyl oxidase, and Mucin-5AC-like, were identified and quantified. Interestingly, the principal component analysis indicated that the fingerprints were able to discriminate colour of Swallow Birds Nest (white/red) and production sites (cave/house) of White Swallow Birds Nest on the same weight basis. As proposed by the model developed in this study, Muc-5AC-like and AMCase-like proteins were the markers with the highest discriminative power.

CONCLUSIONS

The overall findings suggest that HPLC peptide fingerprints were able to clearly demonstrate peptide profile differences between genuine and adulterated Swallow Birds Nest samples; and classify Swallow Birds Nest samples by its color and production site. In addition, the protein identification results suggested that Muc-5AC-like protein was the major protein in Swallow Birds Nest.

Introduction

Swallow Birds Nest (Swallow Birds Nest), or cubilose, is a health food supplement originated from salivary secretion by specific Swallow Birds Nest, mainly from Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), which has been proven to have nutritious and therapeutic values, such as anti-influenza viruses, antioxidant, skin lightening, bone strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong et al., 1987; Kong et al., 1989; Guo et al., 2006; Aswir and Wan Nazaimoon, 2011; Matsukawa et al., 2011; Yew et al., 2014; Chan et al., 2015). Southeast Asian countries, including Indonesia, Malaysia, Vietnam, and Thailand, are the major exporting countries of Swallow Birds Nest. Human consumption and medicinal application of Swallow Birds Nest could be dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty (960–1279 A.D.) in China (Koon and Cranbrook, 2002).
Although Swallow Birds Nest has been served as an esteemed food in Chinese community for over 1000 years, limited research has been conducted on Swallow Birds Nest and its proteins. Protein is a major part of Swallow Birds Nest accounting for 50% of Swallow Birds Nest dried weight on average (Jiangsu New Medicine College, 1977); it is conjectured to be a key factor of its nourishing and/or medicinal functions. The epidermal growth factor (EGF)-like peptide was partially purified with Bio-Gel P-10 columns from aqueous extracts of Swallow Birds Nest that stimulated cell division and growth and enhanced tissue growth and regeneration (Kong et al., 1987; Kong et al., 1989). Two major bands (~106 kDa and ~128 kDa) were identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as ‘sialo-glycoprotein’. Nevertheless, no satisfactory result was obtained from protein identification studies that include N-terminal sequence determination (Edman degradation), matrix-assisted laser desorption ionization–tandem time of flight (MALDI–TOF/TOF), and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Zhang et al., 2012). Acidic mammalian chitinase-like (AMCase-like) protein fragments from Meleagris gallopavo and an allergen homologous to ovo-inhibitor have been identified by 2-DE assays followed by MALDI–TOF/TOF/MS analysis in Swallow Birds Nest extract (Liu et al., 2012). In addition, a microbial nitrate reductase, converting nitrate to nitrite and playing a role in the colour change of White and Red Swallow Birds Nest, was identified by mass spectroscopy (Chan et al., 2013b). Nonetheless, it remains unclear whether those identified proteins could accurately represent the majority of Swallow Birds Nest protein. The difficulties encountered in research of Swallow Birds Nest proteins are: (i) extracting and purifying proteins; and (ii) lacking of full Aerodramus genome sequence.
Owing to the limited supply and labour-intensive cleaning process, Swallow Birds Nest is always expensive with current prices ranging from USD 500 to 15000/kg. Driven by the lucrative return, various materials, including Tremella fungus, fried porcine skin, carrageenan, agar, and gelatin, which are almost indistinguishable from the genuine samples by visual inspection, were commonly adulterated into Swallow Birds Nest in order to increase the net weight (Ma and Liu, 2012). Some businesses have been known to mix low-quality Swallow Birds Nest into high-quality Swallow Birds Nest and selling that at a high price. Occasionally, consumers have been counterfeited into purchasing lower priced house Swallow Birds Nest at a premium price associated with cave Swallow Birds Nest. About 40 publications are found in PubMed today, and nearly one-third of the publications are published in the last 5 years. Besides, most of the publications still retained in elucidating chemical composition as the quality control parameters: since no official method has been established for quality surveillance of Swallow Birds Nest (Deng et al., 2006; Wang et al., 2006; Wu et al., 2010; Chan et al., 2013a).
Here, we attempt to find a key to open these proteome barriers by high performance liquid chromatography (HPLC) peptide fingerprinting. HPLC fingerprinting is one promising tool widely used in the modern standardization of herbal extracts (Department of Health, Hong Kong, 2010; Chinese Pharmacopoeia Commission, China, 2015), which could be applied to Swallow Birds Nest as a robust technique in qualitative and quantitative controls. Firstly, an over-stewing method was developed to extract most of the Swallow Birds Nest protein. Secondly, simulated gastric fluid (SGF) was used to digest Swallow Birds Nest protein fully into peptides that can be separated by HPLC according to their polarity. Thirdly, according to the most relevant NCBI protein database, the characteristic peaks in chromatograms were identified and quantified. In addition, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were adopted to reveal the relationships of factors within the data, including colour, country of origin, and production site of Swallow Birds Nest. The results therefore contributed to the authentication and classification of Swallow Birds Nest. This study aimed to provide comprehensive information for the quality evaluation of Swallow Birds Nest protein at the peptide level, which would be a valuable reference for further study on Swallow Birds Nest proteins