Chinese Herbal Medicines 15 (2023) 383–390

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Chinese Herbal Medicines

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Review

Proteins: Neglected active ingredients in edible bird’s nest

Man Yuan a, Xiaoxian Lin a, Dongliang Wang a,b,⇑, Jianye Dai a,b,⇑
a
Hebei Edible Bird’s Nest Fresh Stew Technology Innovation Center, Langfang 065700, China
b
School of Pharmacy, Lanzhou University, Lanzhou 730000, China

a r t i c l e i n f o a b s t r a c t

Article history: Edible bird’s nest (EBN) is a kind of natural invigorant with a long history of consumption in Asia, espe-
Received 8 November 2022 cially in China. EBN is formed by mixing the saliva of swiftlets (Aerodramus) with feathers and other com-
Revised 15 January 2023 ponents during the breeding season. Proteins are the most important nutrient in EBN. By studying
Accepted 21 February 2023
proteins in EBN, we can not only elucidate their components at the molecular level, but also study their
Available online 16 May 2023
bioactivities. Therefore, it is of great significance to study the proteins in EBN. Previous research on the
proteins in EBN was preliminary and cursory, and no one has summarized and analyzed the proteins
Keywords:
in EBN and correlated the bioactivities of these proteins with the biological functions of EBN. This article
acidic mammalian chitinase
bioactivity
focused on the proteins in EBN, listed the proteins identified in different proteomic studies, and intro-
edible bird’s nest duced the sources, structures and bioactivities of the most frequently identified proteins, including acidic
lysyl oxidase homolog 3 mammalian chitinase, lysyl oxidase homolog 3, mucin-5AC, ovoinhibitor, nucleobindin-2, calcium-
mucin-5AC binding protein (MW: 4.5  104) and glucose-regulated protein (MW: 7.8  104). The properties of these
proteins proteins are closely related to the bioactivities of EBN. Therefore, this article can provide inspiration for
further research on the efficacy of EBN.
Ó 2023 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V. This is an open access article
under the CC BY-NC-ND license ([Link]

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
2. Proteins identified in EBN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3. Main proteins of EBN and their bioactivities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3.1. Acidic mammalian chitinase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
3.2. Lysyl oxidase homolog 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
3.3. Mucin-5AC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
3.4. Ovoinhibitor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
3.5. Nucleobindin-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
3.6. Calcium-binding protein (MW: 4.5  104) and glucose-regulated protein (MW: 7.8  104). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
4. Discussion and prospective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

1. Introduction with their feathers and other substances for laying eggs and incu-
bation during the breeding season (Shao et al., 2018, Fig. 1A). When
Edible bird’s nest (EBN) is a kind of natural invigorant formed the baby swiftlets fly away as an adult, the nest will be abandoned,
by the saliva secreted by several species of swiftlets (Aerodramus) then it will be manually collected and processed into EBN. In China,
EBN has always been regarded as premium food and invigorant,
and it was known as the ‘‘Caviar of the East” (Marcone, 2005;
⇑ Corresponding authors.
Jian et al., 2016a). In the past, EBN has always been the favorite
E-mail addresses: [Link]@[Link] (D. Wang), daijy@[Link]
(J. Dai).
food of the royal family and nobles. Nowadays, EBN is also

[Link]
1674-6384/Ó 2023 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V.
This is an open access article under the CC BY-NC-ND license ([Link]
M. Yuan, X. Lin, D. Wang et al. Chinese Herbal Medicines 15 (2023) 383–390

Fig. 1. Swiftlet and EBN (Original photo by Yingzhi Fu). A, swiftlet and nest; B, dried EBN (raw material); C, fresh stewed EBN.

regarded as premium food for important occasions such as state more researchers had carried out the analysis and identification
banquets and diplomacy. Besides, the efficacy and medical value of EBN proteins by different methods such as GPC, RP-HPLC, etc.
of EBN were also recorded in more than 20 classics of Chinese Med- Statistical analysis showed that a total of 58 proteins have been
icine. According to the Essential of Materia Medica (”Ben Cao Bei identified in previous studies (Table 1).
Yao‘‘, in Chinese), EBN can greatly nourish the lung yin, relieve
phlegm and cough, gently nourish the body, and is very effective
3. Main proteins of EBN and their bioactivities
medicine to regulate fatigue. Modern researches have also shown
that EBN has the functions of immune regulation (Zhao et al.,
There were seven kinds of proteins that have been identified
2016), anti-aging (Yew, Koh, Chye, Othman, & Ng, 2014), and reg-
three times or more in different EBN protein analysis and identifi-
ulation of intestinal flora (Bai, Liu, Zhang, Fan, & Xu, 2020). There-
cation, including acidic mammalian chitinase, lysyl oxidase homo-
fore, EBN has an important position in premium food and
log 3, mucin-5AC, ovoinhibitor, nucleobindin-2, calcium-binding
invigorant in China. The market size of EBN in China is about 50–
protein (MW: 4.5  104) and glucose-regulated protein (MW:
60 billion yuan per year, and the common product types are dried
7.8  104). The times of identification and biological activities of
EBN (Fig. 1B) and instant EBN represented by fresh stewed EBN
these seven proteins have been summarized (Table 2).
(Fig. 1C).
Different sources, types, picking seasons and other factors of
EBN may lead to differences in the chemical composition. Mod- 3.1. Acidic mammalian chitinase
ern research shows that, nutrients of EBN mainly include pro-
teins, sialic acid, amino acids, vitamins, inorganic elements, etc. Acidic mammalian chitinase (AMCase, Fig. 2A) is a glycosidase
Among them, sialic acid has been considered as the main nutri- that belongs to the glycosyl hydrolase family 18, the chitinase class
ent in EBN, but its content only accounts for about 10% of the II subfamily. It is expressed in the cytoplasm of most tissues, highly
total mass (Jian et al., 2016b). However, the protein which expressed in the stomach, and also detected in the lung. A protein
accounts for 50% 67% (Bai, Liu, Zhang, Fan, & Xu, 2020) of the that was highly abundant in all 15 EBN samples had been discov-
total mass of EBN has always been neglected. But in fact, pro- ered through MALDI-TOF-TOF/MS (Liu et al., 2012), and it was
teins are the main nutrient in many animal-derived foods, such identified as an AMCase-like protein fragment derived from the
as EBN and Colla CoriiAsini. The authenticity can be identified purslane. Chitin is an important component in structures such as
based on the protein composition, while the bioactivities of fungal cell walls and insect exoskeletons. Chitinase can degrade
these food can also be studied according to these proteins. chitin components, and AMCase-like protein is highly expressed
Therefore, it is of great significance to identify and study the in the salivary gland of the swiftlets to secrete saliva for insect
proteins contained in EBN. digestion (Yew et al., 2019).
In recent years, some studies have preliminarily identified the AMCase plays an important role in antiviral, anti-inflammatory
proteins in EBN. Through the study of these proteins, it is found and cell protection from apoptosis. The study found that AMCase
that many of them (such as acidic mammalian chitinase) play identified in EBN was involved in resisting the invasion of nema-
important biological activities in human body, such as immune todes, fungi and other chitin containing pathogens (Zukefli, Chua,
regulation. However, no one has systematically summarized these & Rahmat, 2017). It can control the inflammatory response through
proteins and their biological activities. Therefore, this paper lists IL-13, the main regulator of th2 immune response (Hartl et al.,
the EBN proteins that have been identified so far, and summarizes 2008), which can be used to explain the anti-inflammatory proper-
the sources, structures and biological activities of the most com- ties and the inhibition of influenza virus activity of EBN (Mohamad,
monly identified proteins in order to provide new ideas for the bio- Mohamad, Abu, Mahmud, & Razak, 2021).
logical activity research of EBN. In addition, chitin can promote the development of lung dis-
eases. It has been reported that the chitin polymers in the airways
of mice lacking AMCase accumulated continuously, stimulated the
2. Proteins identified in EBN expression of fibrogenic cytokines, and developed spontaneous
pulmonary fibrosis (Van et al., 2017). More importantly, these
Previous studies of the proteins in EBN were preliminary and symptoms can be improved by restoring AMCase activity in the
cursory. Few people have analyzed and identified exactly which lungs of these mice. It can be seen that AMCase can prevent or alle-
proteins are contained in EBN. In recent years, as a powerful tool viate pulmonary diseases such as pulmonary fibrosis by decompos-
to identify proteins, proteomics played an important role in the ing chitin.
study of EBN proteins. Several studies have attempted to correlate In addition to the above functions, AMCase can also stimulate
EBN properties or biological functions with its protein composition pulmonary epithelial cells to produce chemokines and protect
through proteomic methods. them from apoptosis (Hartl et al, 2008; Hartl et al, 2009), which
Liu et al. (2012) first used MALDI-TOF-MS in 2012 to analyzed explained the epidermal growth factor (EGF) like effects found in
the protein composition in 15 EBN samples. Since then, more and early studies of EBN. It is worth noting that the anti-
384
M. Yuan, X. Lin, D. Wang et al. Chinese Herbal Medicines 15 (2023) 383–390

Table 1
EBN proteins that have been identified.

Proteins References
Acidic mammalian chitinase Kong, Wong, & Lo, 2016; Liu et al., 2012; Ma et al., 2019; Mohamad, Mohamad, Abu, Mahmud, & Razak,
2021; Wong et al., 2017; Wong et al., 2018; Yew et al., 2019; You et al., 2015; Zukefli, Chua, & Rahmat, 2017
Actin, cytoplasmic type 5 Ma et al., 2019
Actin, gamma-enteric smooth muscle Ma et al., 2019
BEN (bursal epithelium and neurons) glycoprotein Zukefli, Chua, & Rahmat, 2017
precursor
Collagen alpha-2(I) chain Yew et al., 2019; Zukefli, Chua, & Rahmat, 2017
Collagen alpha-1(VII) chain-like Zukefli, Chua, & Rahmat, 2017
Carbonic anhydrase 9 Kong, Wong, & Lo, 2016
Coenzyme Q-binding protein COQ10 homolog A, Zukefli, Chua, & Rahmat, 2017
mitochondrial, partial
Carboxypeptidase Zukefli, Chua, & Rahmat, 2017
Calcineurin-like phosphoesterase domain containing Yew et al., 2019
protein 1
Cytochrome b Ghassem, Arihara, Mohammadi, Sani, & Babji, 2017
Deleted in malignant brain tumors 1 protein Ma et al., 2019; Yew et al., 2019
Fibronectin type III domain-containing protein 3B Yew et al., 2019
Glycosyltransferases Ghassem, Arihara, Mohammadi, Sani, & Babji, 2017
Hyaluronan mediated motility receptor Yew et al., 2019
Hemojuvelin Ma et al., 2019
Integrator complex subunit 1 Yew et al., 2019
Immunoglobulin superfamily member 10-like, partial Kong, Wong, & Lo, 2016
Ig heavy chain V-III region VH26, partial Kong, Wong, & Lo, 2016
IgL, partial Kong, Wong, & Lo, 2016
Keratin, type II cytoskeletal 75 Yew et al., 2019
Ma et al., 2019; Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Wong et al., 2017; Wong et al., 2018;
Lysyl oxidase homolog 3 Yew et al., 2019; Zukefli, Chua, & Rahmat, 2017
Lysosomal-trafficking regulator Yew et al., 2019
Leucine-rich repeat-containing protein 9 Zukefli, Chua, & Rahmat, 2017
Kong, Wong, & Lo, 2016; Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Wong et al., 2017; Wong
Mucin-5AC et al., 2018; Yew et al., 2019
Mucin-1 Kong, Wong, & Lo, 2016
Mucin-2 Kong, Wong, & Lo, 2016
Mucin-19-like Kong, Wong, & Lo, 2016
Mucin-22-like Kong, Wong, & Lo, 2016
Major facilitator superfamily domain containing 11 Zukefli, Chua, & Rahmat, 2017
isoform X4
Nucleobindin-2 Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Wong et al., 2018; Yew et al., 2019
Nucleosome assembly protein 1-like 4 Zukefli, Chua, & Rahmat, 2017
NADH dehydrogenase subunit 2 Kong, Wong, & Lo, 2016
Neuropilin and tolloid-like 1 Yew et al., 2019
Non-specific lipid-transfer protein isoform X5 Zukefli, Chua, & Rahmat, 2017
Ovoinhibitor Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Wong et al., 2018; Yew et al., 2019
Ovotransferrin precursor Ghassem, Arihara, Mohammadi, Sani, & Babji, 2017
Protein arginine N-methyltransferase7 Zukefli, Chua, & Rahmat, 2017
Putative threonine–tRNA ligase 2, cytoplasmic Yew et al., 2019
Protein lin-9 homolog Yew et al., 2019
Protein bassoon Yew et al., 2019
PDZ domain-containing protein GIPC3 isoform X3 Zukefli, Chua, & Rahmat, 2017
Pre-rRNA-processing protein TSR1 homolog isoform X3 Zukefli, Chua, & Rahmat, 2017
Peroxisomal membrane protein PEX16 Zukefli, Chua, & Rahmat, 2017
RGM domain family member B Yew et al., 2019; Zukefli, Chua, & Rahmat, 2017
Receptor-type tyrosine-protein phosphatase gamma Zukefli, Chua, & Rahmat, 2017
SANT domain DNA-binding protein Zukefli, Chua, & Rahmat, 2017
Selenocysteine-specific elongation factor Yew et al., 2019
Signal transducer and activator of transcription 6-like Zukefli, Chua, & Rahmat, 2017
isoform X5
Small proline-rich protein 3, partial Kong, Wong, & Lo, 2016
Sushi, von Willebrand factor type A, EGF and pentraxin Kong, Wong, & Lo, 2016
domain-containing protein 1, partial
Transmembrane protein 186 Zukefli, Chua, & Rahmat, 2017
Von Willebrand factor D and EGF domain-containing Kong, Wong, & Lo, 2016
protein
WD repeat-containing protein 72 isoform X5 Zukefli, Chua, & Rahmat, 2017
Zona pellucida sperm-binding protein 2 Yew et al., 2019
ZZ-type zinc finger-containing protein 3 isoform X5 Zukefli, Chua, & Rahmat, 2017
Calcium-binding protein (MW: 4.5  104) Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Wong et al., 2018; Yew et al., 2019
Glucose-regulated protein (MW: 7.8  104) Mohamad, Mohamad, Abu, Mahmud, & Razak, 2021; Yew et al., 2019; Zukefli, Chua, & Rahmat, 2017

inflammatory and cytoprotective functions of AMCase are 3.2. Lysyl oxidase homolog 3
inhibited by allosamidin, but this inhibition will not affect the
chitinolytic activity of AMCase, which means that these functions Lysyl oxidase (LOX) is a copper-dependent amine oxidase
of AMCase may not be related to its chitinase activity. responsible for the formation of lysine-derived crosslinks in extra-

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Table 2 nificantly improved compared with other groups (Matsukawa

Main proteins of EBN and their bioactivities. et al., 2011). Besides, the use of LOX family inhibitors b-APN led
Proteins Bioactivities Number of to an increase in arterioscleroserosis lesions, suggesting that
times LOXL3 plays an important role in the integrity of the blood vessel
identified walls (Remus et al., 2012). In addition, research has found that
Acidic mammalian Degrade chitin. Play a role in antiviral, 9 LOXL3 enhanced integrin signaling in muscle fibers and its adhe-
chitinase anti-inflammatory and cell protection sion to tendon junctions by catalyzing the oxidation of fibronectin
from apoptosis. Control the Th2
inflammatory response through IL-13.
during development {Kraft-Sheleg, 2016 #37}(Kraft et al., 2016).
Prevent or alleviate pulmonary Moreover, a novel enzymatic activity of LOXL3 in removing the
diseases. Stimulate pulmonary acetyl and acetamido groups of STAT3 has been reported, which
epithelial cells to produce chemokines is helpful to control the inflammatory reaction and proved the
and protect them from apoptosis.
physiological role of LOXL3 in immune regulation (Ma et al.,
Lysyl oxidase Indispensable in normal development. 6
homolog 3 Essential for stabilizing fibril and 2017). The above studies indicated that LOXL3 played an impor-
elastin elasticity. Play a role in the tant role in maintaining the health of skin, cardiovascular, tendon
integrity of the blood vessel walls. and immune system.
Enhance integrin signaling in muscle In addition to the above functions, LOXL3 also plays a role in the
fibers and its adhesion to tendon
pathogenesis of diseases such as Stickler syndrome, Keratoconus,
junctions by catalyzing the oxidation
of fibronectin during development. and glaucoma (Sethi, Mao, Wordinger, & Clark, 2011). Up-
Remove the acetyl and acetamido regulated expression of LOXL3 was detected in the ciliated bron-
groups of STAT3 which is helpful to chial epithelium of lung fibrotic tissue compared to control lung
control the inflammatory reaction and
tissue (Jones et al., 2018; Aumiller et al., 2017; Chen, Li, & Li,
immune regulation.
Mucin-5AC Create a tight gel that acts as a 5 2019). In addition, all LOX expression levels were elevated in the
protector. Mediate the formation of a lungs of patients with idiopathic pulmonary hypertension (Nave
bicarbonate gradient and can form a et al., 2014). It has been reported that in systemic hypertension,
bilayer mucus barrier together with elevated expression of LOXL3 was observed in cardiac fibroblasts
MUC6 that protect epithelial cells from
and cardiomyocytes (Yu, Horak, & Larson, 2006). LOXL3 is also
the deleterious effects of luminal acids.
Soluble in tear fluid for the capture and one of the genes up-regulated in microarray studies of affected car-
removal of foreign debris. Prevent tilage in osteoarthritis (Yu, Horak, & Larson, 2006). Huang et al.
virus and parasites. Defense against (2016) also confirmed that LOXL3 is involved in osteoarthritis. In
some intestinal parasites that invade
addition, LOXL3 is also closely related to a variety of tumors and
the lungs.
Ovoinhibitor Involve in antibacterial defense by 3
cancers, and its expression was found in myeloproliferative tumors
inhibiting the microbial proteases that (Tadmor et al., 2013), breast cancer, ovarian cancer (Sebban,
constitute the major components. Davidson, & Reich, 2009), and colorectal cancer (Barbazán et al.,
Prevent the shedding of rotavirus 2014; Insua et al., 2017), etc. Research has shown that LOXL3 is
antigens and delay the development of
involved in protein interactions in tumor progression and metasta-
rotavirus gastroenteritis. May be the
main cause of IgE allergic reactions. sis as well as genome integrity (Peinado et al., 2005). The above
Nucleobindin-2 Play a role in preventing insulin 3 findings suggested that LOXL3 may be a potential therapeutic tar-
resistance, maintaining calcium get for a variety of diseases, but its role in many pathological mech-
homeostasis. Promot cell proliferation
anisms remains to be further studied and verified.
and inhibit adipocyte differentiation.
Calcium-binding Alleviate endoplasmic reticulum 3
protein (MW: stress. Involve in regulating calcium- 3.3. Mucin-5AC
4.5  104) dependent activities in the lumen or
posterior compartment of the Mucin is a family of high molecular weight, heavily glycosylated
endoplasmic reticulum, which may
large glycoproteins that are commonly found in saliva and breast
have an essential function in cellular
calcium homeostasis. milk. Mucin-5AC (MUC5AC, Fig. 2C) is a secreted, gel-forming
Glucose-regulated Alleviate endoplasmic reticulum 3 mucin, mainly expressed in the stomach, eyes, and airways. The
protein (MW: stress. Anti-apoptosis. May contribute highly glycosylated MUC5AC, by virtue of its cysteine-rich repeat
7.8  104) to neuroprotective effect. Prevent the
sequence, can form intermolecular and intramolecular disulfide
transport of misfolded proteins or
protein subunits.
bonds, resulting in complex polymers that form a framework of
polymerized mucus gels on the surface of epithelial cells. Gel-
forming mucins are important components of the mucus layer
and function as an epithelial cell barrier that coats and protects
cellular matrix proteins such as collagen and elastin. Lysyl oxidase the epithelial surface from environmental aggression and prevents
homolog 3 (LOXL3, Fig. 2B), a member of the lysyl oxidase family, is the invasion of pathogens (Krishn, Ganguly, Kaur, & Batra, 2018;
highly expressed in the heart, spleen, uterus, lung, aorta, and coro- Okuda et al., 2019). Gastrointestinal digestion of EBN was simu-
nary arteries, and also expressed in the corneal layer, trabecular lated in vitro and the protein (MW: 5.0  104) in EBN was
meshwork, limbus, and conjunctiva (Battle, Brown, Engelhardt, & sequenced using in-gel digestion combined with MALDI-TOF-
Montgomery, 2017; Collaborators, 2013; Dudakova, Sasaki, TOF/MS, which was found to match multiple mucin sequences
Liskova, Palos, & Jirsova, 2016; Melé et al., 2015). It has been found from different birds. And the presence of MUC5AC in EBN was fur-
that LOXL3-deficient mice exhibited reduced thoracic cavity and ther confirmed by Western blot experiments using mucin antibody
pulmonary hypoplasia, indicated that LOXL3 is indispensable in (Zukefli, Chua, & Rahmat, 2017).
normal development (Zhang et al., 2016). Recent studies of LOXL3 MUC5AC plays a very important role in the health of the stom-
have revealed the role beyond its canonical function as an amino ach and eyes, and its unique characteristics allow it to create a
oxidase. tight gel that acts as a protector. In the stomach, MUC5AC mediates
LOXL3 is essential for stabilizing fibril and elastin elasticity. the formation of a bicarbonate gradient and can form a bilayer
The skin of ovariectomized rats supplemented with EBN was sig- mucus barrier together with mucin-6 (MUC6) that protects epithe-
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M. Yuan, X. Lin, D. Wang et al. Chinese Herbal Medicines 15 (2023) 383–390

Fig. 2. 3D structures of seven main EBN proteins (Jumper et al., 2021). A: 3D structure of acidic mammalian chitinase (AF-Q9BZP6-F1); B: 3D structure of lysyl oxidase
homolog 3 (AF-P58215-F1); C: 3D structure of mucin-5AC (AF-Q14887-F1); D: 3D structure of ovoinhibitor (AF-P10184-F1); E: 3D structure of nucleobindin-2 (AF-P80303-
F1); F: 3D structure of 4.5  104 calcium-binding protein (AF-Q9BRK5-F1); G: 3D structure of 7.8  104 glucose-regulated protein (AF-A0A7P0TAI0-F1).

lial cells from the deleterious effects of luminal acids (Ermund, role of MUC5AC in these pathological mechanisms remains to be
Schütte, Johansson, Gustafsson, & Hansson, 2013). These findings further studied and verified.
suggested that the role of EBN in the treatment and prevention
of gastric diseases may be derived from MUC5AC.
Mucin loss of the tear film and ocular glycocalyx is thought to 3.4. Ovoinhibitor
be the core pathogenesis of dry eye, and the key point of this mech-
anism is hyperosmolarity of tear fluid caused by high evaporation Ovoinhibitor (Fig. 2D) is a serine protease inhibitor belonging to
or low tear flow. Hypertonic tear fluid can lead to tear film damage, the Kazal family that specifically inhibits serine proteases such as
inflammatory response, and loss of surface mucin, which in turn trypsin, chymotrypsin, subtilisin, and porcine elastase (Shechter,
can lead to tear film instability. Mucin is an important component Burnstein, & Gertler, 1977; Matsuda, Watanabe, & Nakamura,
of the tear film and the apical surface of the cornea, and the main 1983). Ovoinhibitor was originally purified from egg white
mucin expressed by the cup cells of the conjunctiva is MUC5AC. In (Lineweaver & Murray, 1947) and is highly expressed in the large
addition, MUC5AC was shown to be soluble in tear fluid, which in intestine, liver and uterus of birds, responsible for the secretion
turn moved the soluble mucin to the surface of the eye for the cap- of egg white, yolk and eggshell precursors, respectively. Since the
ture and removal of foreign debris (Argüeso, Spurr, Russo, Tisdale, EBN is the swiftlet’s nest for laying eggs, the ovoinhibitor in EBN
& Gipson, 2003; Gipson, Hori, & Argüeso, 2004). may come from the swiftlet’s eggs (Wong et al., 2018).
Some previous studies have suggested that mucus formed by Ovoinhibitor may be involved in antibacterial defense by
MUC5AC may adversely affect asthma and chronic muco- inhibiting the microbial proteases that constitute the major com-
obstructive pulmonary diseases, and proposed to treat these dis- ponents. Studies have shown that, purified ovoinhibitor was
eases by inhibiting MUC5AC. But this opinion may be one-sided. shown to inhibit trypsin, Bacillus subtilis, Bacillus thuringiensis,
Mucins are part of the lung’s innate defense system. In the respira- and Staphylococcus aureus (Bourin et al., 2011). The above results
tory system, MUC5AC is mainly secreted by surface cells of the lar- indicated that ovoinhibitor played an important role in preventing
ger airways. However, the production of MUC5AC is regulated by contamination of unfertilized eggs and protecting fertilized chick
the type 1 (viral infections and cigarette smoke) and type 2 (aller- embryos. Research has found that ovoinhibitor could prevent the
gens) immune responses, which makes the small airways also shedding of rotavirus antigens and delay the development of rota-
begin to secrete MUC5AC. And this is why it is closely related to virus gastroenteritis in mice model of rotavirus infection (Yolken,
asthma and chronic muco-obstructive pulmonary diseases Willoughby, Wee, Miskuff, & Vonderfecht, 1987). In addition, it is
(Kesimer et al., 2017; Radicioni et al., 2021). It is speculated that worth noting that, ovoinhibitor in EBN may be the main cause of
MUC5AC may plays a similar protective role in the lungs as in IgE allergic reactions, especially in children (Goh, Chew, Chua,
the stomach. The tight gel formed by MUC5AC facilitates isolation Chay, & Lee, 2000).
of acute lesions and prevents the migration of invasive pathogens, Serine protease inhibitors in humans are highly expressed in
including viruses, through the lung to other sites. MUC5AC is the thymus and stratum corneum, and are also found in the oral
known to prevent virus that can induce type I inflammation mucosa, parathyroid, bartholin, tonsil, and vaginal epithelium,
(Hewson et al., 2010) and parasites that can induce type II inflam- with very low levels detected in the lung, kidney, and prostate
mation (Hasnain et al., 2011), it also critical for defense against (Mägert et al., 1999). Serine protease inhibitors in humans con-
some intestinal parasites that invade the lungs (Hasnain et al., tribute to skin integrity and protective barrier function by modu-
2011). Therefore, it is unwise to blindly treat asthma and chronic lating defense activation and the activity of desquamation-
muco-obstructive pulmonary diseases by inhibiting MUC5AC. The related protease, which are important for anti-inflammatory and
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antimicrobial protection of the mucous epithelium (Deraison et al., GRP78 prevents the transport of misfolded proteins or protein sub-
2007). units. Under ER stress conditions, when unfolded proteins accumu-
late in the ER lumen, GRP78 activates the UPR and alleviates ER
3.5. Nucleobindin-2 stress by reducing protein translation and increasing ER folding
capacity (Aridon et al., 2011; Leak, 2014).
Nucleobindin-2 (NUCB2, Fig. 2E) is a calcium-binding protein
that was originally identified in the acute lymphoblastic leukemia 4. Discussion and prospective
cell line KM3 (Barnikol et al., 1994). Nucleonectin-2 is expressed in
the cytoplasm of most tissues including immune cells, mainly in Numerous studies have demonstrated the health benefits of
the spleen, testis and stomach. It response to endoplasmic reticu- EBN, many of which coincide with the bioactivities of proteins
lum (ER) stress, while activating transcription factor 6 (ATF6) is identified in EBN, further evidencing that some of the bioactivities
transported to the Golgi and cleaved by site 1 protease (S1P) of EBN are likely derived from these proteins.
(Tsukumo et al., 2007). Nucleonectin-2 plays a role in preventing Immune regulation is one of the most studied bioactivities of
insulin resistance, maintaining calcium homeostasis, promoting EBN. The human immune system consists of three lines of defense,
cell proliferation and inhibiting adipocyte differentiation. and various proteins in EBN participate in the immunity through
Nucleonectin-2 binds to adrenaline to maintain calcium home- different mechanisms. The first line of immune defense consists
ostasis. It is post-translationally processed into the biologically of the skin, mucous membranes and their secretions. The skin cov-
active nesfatin-1 peptide, which has been reported to induce sati- ers the surface of the body extensively, keeping pathogens such as
ety, thereby increasing insulin sensitivity. Studies have shown that, bacteria and viruses out of the body. While the mucous membrane
loss of nucleonectin-2 exacerbates metabolic inflammation in adi- mainly covers the oral and nasal cavity, digestive tract, alveoli and
pose tissue macrophages in an NF-jB-dependent manner when fed other parts, and the mucus and bactericidal substances secreted by
a high-fat diet (Ravussin et al., 2018). The results showed that dele- it can block, capture and remove foreign substances. AMCase and
tion of nucleonectin-2 did not affect food intake or cause weight ovoinhibitors in EBN help maintain skin integrity, while MUC5AC
gain, but instead caused metabolic inflammation and insulin resis- and ovoinhibitors are involved in the antimicrobial protection of
tance in mice fed a high-fat diet. It can be seen that nucleonectin-2 mucous epithelium. The second line of immune defense is humoral
plays a regulatory role in insulin secretion and can be a potential immunity, including bactericidal substances and phagocytes in
contributor to the study of diabetes pathology. body fluids. The AMCase in EBN plays a role in humoral immunity
Furthermore, nucleonectin-2 is a positive regulator of EGF- by activating Th2 cells to produce IL-13 that can control the anti-
dependent signaling, and it can enhance cell growth and inhibit inflammatory response. The first two lines of defense are non-
adipocyte differentiation. It has been reported that endogenous specific immunity, which is not directed against a specific patho-
nucleonectin-2 inhibited PP2A activity by controlling intracellular gen but has a defensive effect against a variety of pathogens. The
free calcium concentration, thereby enhancing EGF activation of third line of defense is specific immunity, which only acts against
MEK/Erk modules and promoting cell proliferation (Yoshikawa a specific pathogen, and is mainly composed of immune organs
et al., 2000). Therefore, extracellular signal regulated kinases and immune cells with the help of blood circulation and lymphatic
(ERK) activity most likely explains the ability of nucleonectin-2 circulation. LOXL3 in EBN helps control inflammatory responses by
to enhance cell growth (Ramos, 2008). In addition, the study also removing the acetyl and acetamido groups of STAT3, while both
found that nucleonectin-2 had the function of adipose suppressor, SDF4 and GRP78 can exert anti-inflammatory effects by alleviating
and its high expression can inhibit the differentiation of 3T3-L1 ER stress.
adipocytes. In summary, it is suggested that the EGF effect and In addition to the above-mentioned immune regulation effects,
potential weight loss function of EBN may be closely related to other bioactivities of EBN are also closely related to the proteins in
nucleonectin-2. it. For example, in addition to the sialic acid and EGF, the effect of
EBN in maintaining skin elasticity and anti-aging may also benefit
3.6. Calcium-binding protein (MW: 4.5  104) and glucose-regulated from the stabilizing effect of LOXL3 on fibrils and elastin in the
protein (MW: 7.8  104) skin, and the role of nucleobindin-2 in promoting cell proliferation.
While the protective effect of EBN on the stomach and lungs may
Mohamad et al. {Mohamad Nasir, 2021 #24}(2021) have found be derived from the protection of AMCase and MUC5AC on lung
4.5  104 calcium-binding protein (SDF4, Fig. 2F) and 7.8  104 epithelium and gastric mucosa. In addition, the other functions of
glucose-regulated protein (GRP78, Fig. 2G) in raw EBN and its EBN including protective effects on nervous system, cardiovascu-
aqueous extracts. Both proteins can alleviate ER stress, which is lar, tendon, bone and joint, eyes, and prevention of diabetes and
closely related to the pathological mechanism of immune- obesity, maintenance of cellular calcium homeostasis, etc. are clo-
inflammatory diseases. Therefore, these two proteins may have sely related to the bioactivities of MUC5AC, ncleobindin-2, SDF4,
anti-inflammatory effect. GRP78, etc.
SDF4 is composed of 361 amino acids and is present in brain It should be noted that, although the proteins mentioned in this
regions in astrocytes, neurons, oligodendrocyte progenitors, myeli- article were identified in EBN, it does not mean that they still have
nated oligodendrocytes, microglia, macrophages and highly their original bioactivities after being digested and absorbed by the
expressed in endothelial cells. Studies have shown that the expres- human body. Whether the peptides obtained after the digestion of
sion of SDF4 can attenuate ER stress. And it is involved in regulat- EBN also have similar bioactivities still needs to be studied through
ing calcium-dependent activities in the lumen or posterior more pharmacological experiments. In addition, the protein analy-
compartment of the ER, which may have an essential function in sis of EBN is affected by different extraction methods, and factors
cellular calcium homeostasis (Zhu et al., 2021). such as the source, type, and picking season of EBN may also lead
GRP78 is a member of the heat shock protein 70 family, exists to differences in its chemical composition, including the identified
on the ER membrane and is the main binding protein for Ca2+. protein components. And there may still be many proteins in EBN
GRP78 has anti-apoptotic effects (Ibrahim, Abdelmalek, & Elfiky, that have not been identified by analysis. Therefore, further explo-
2019; Pfaffenbach & Lee, 2011) and may contribute to neuropro- ration of the known bioactivities of EBN and the association of pro-
tection in the 6-OHDA-treated neuroblastoma cell model SH- teins, as well as the unknown potential bioactivities, requires more
SY5Y (Yew, Koh, Chye, Othman, & Ng, 2014). In normal cells, in-depth and detailed research.
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M. Yuan, X. Lin, D. Wang et al. Chinese Herbal Medicines 15 (2023) 383–390

Declaration of Competing Interest Hewson, C. A., Haas, J. J., Bartlett, N. W., Message, S. D., Laza, S. V., Kebadze, T., ...
Johnston, S. L. (2010). Rhinovirus induces MUC5AC in a human infection model
and in vitro via NF-jB and EGFR pathways. European Respiratory Journal, 36(6),
The authors declare that they have no known competing finan- 1425–1435.
cial interests or personal relationships that could have appeared Huang, Z. M., Du, S. H., Huang, L. G., Li, J. H., Xiao, L., & Tong, P. (2016). Leptin
promotes apoptosis and inhibits autophagy of chondrocytes through
to influence the work reported in this paper.
upregulating lysyl oxidase-like 3 during osteoarthritis pathogenesis.
Osteoarthritis and Cartilage, 24(7), 1246–1253.
Ibrahim, I. M., Abdelmalek, D. H., & Elfiky, A. A. (2019). GRP78: A cell’s response to
Acknowledgments stress. Life Sciences, 226, 156–163.
Insua, Y. V., Cámara, J., Vázquez, E. B., Fernández, A., Rivera, F. V., Silva, M., ... Alonso,
A. L. (2017). Predicting outcome and therapy response in mCRC patients using
This work is financially supported by the Fundamental Research
an indirect method for CTCs detection by a multigene expression panel: A
Funds for the Central Universities (lzujbky-2022-ct03). multicentric prospective validation study. International Journal of Molecular
The authors are thankful to Yingzhi Fu from Beijing Xiaoxian- Sciences, 18(6), 1265.
Jian, Y. Y., Li, Q. W., Huang, C. C., Huang, Z. J., Xie, Y., Zeng, B. D., & Zang, Y. (2016).
dun Biotechnology Co., Ltd. for providing the EBN photos.
Analysis and comparison of nutrient composition of dried bird’s nest and
stewed bird’s nest. The Light & Textile Industries of Fujian, 3, 32–38.
Jian, Y. Y., Li, Q. W., Huang, Z. J., Huang, C. C., Zheng, L. J., Fu, W. Q., ... Zang, Y. (2016).
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