Bioavailability Evaluation of a New Compound Bone Peptide

Formula

Yupeng Song

, Jiakang Mu

, Chunyang Ding

, Jiamin Xue

, Shiqi Li

, Sa Zhou

Aqin Wang

2,* g

and Jun Yu

2,* h

Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education & Tianjin Key Laboratory of Industrial

Microbiology, College of Biotechnology, Tianjin University of Science and Technology, 300457, Tianjin, China

Hangzhou Bibau Biotechnology Co Ltd., 310016, Hangzhou, China

zhousa@tust.edu.cn, haiziaqin@126.com, yujunsz@163.com

Keywords: Bone Peptide, Hydroxyproline, Collagen Peptide, Bioavailability.

Abstract: To investigate the contents of hydroxyproline (Hyp) and Glycine-Proline- Hydroxyproline (Gly-Pro-Hyp) in

the serum of C57/BL mice after the new compound bone peptide formula was gavaged. The bioavailability

and utilization efficiency of the new compound bone peptide formula were evaluated by the contents of above

substances in the serum. Moreover, the area under the drug time curve (AUC) was analyzed and compared to

explore the effective exposure amount of the new bone peptide formula. The results showed that the contents

of Hyp and Gly-Pro-Hyp increased in a dose-dependent manner in the serum. The contents of Hyp and Gly-

Pro-Hyp were the highest after 3 h and remained in 24 h. The results indicated that the utilization rate of bone

peptide samples was significantly higher than that of bone peptide tablet and bone peptide injection. The AUC

of Hyp and Gly-Pro-Hyp in the low-dose bone peptide sample group were significantly higher than that of

clinical dose bone peptide samples. AUC of Hyp and Gly-Pro-Hyp in the low-dose bone peptide sample group

was significantly higher than those in the clinical dose bone peptide injection and bone peptide tablet groups,

indicating that the clinically dose low-dose bone peptide tablet had a good effective exposure to efficacy.

1 INTRODUCTION

Collagen is one of the main macromolecules that

constitutes the extracellular matrix. Bovine bone

peptide is an active peptide obtained by enzymatic

digestion of bovine collagen, which completely opens

the triple helix structure of bovine collagen, and the

peptide chain is degraded into short peptide chains to

obtain a peptide mixture with a molecular mass of

several thousand daltons.

These small molecule peptides are compatible

with living organisms and have better nutritional

functions than proteins and amino acids (Ahn 2019,

https://orcid.org/0000-0002-8604-5183

https://orcid.org/0000-0001-7093-005X

https://orcid.org/0000-0002-5469-711X

https://orcid.org/0000-0002-4117-3675

https://orcid.org/0000-0001-5979-7171

https://orcid.org/0000-0001-8816-6985

https://orcid.org/0000-0002-6960-5441

https://orcid.org/0000-0003-2402-8000

Bello 2006, Cao 2020, Lee 2019). At the same time,

it has good solubility and stable physical properties.

The small molecular peptides obtained from different

sources and different enzymatic hydrolysis processes

have different biological activities, such as immune

regulation, lowering blood pressure, regulating blood

sugar and blood lipid, anti-aging, anti-oxidation, anti-

cancer, anti-microbial, anti-toxin, increasing bone

density and improving bone toughness (Li, Wu 2018,

Pountos 2016, RosanoBraun 1987). This study

investigated the bioavailability of bovine osteoptin in

mice after administration.

Song, Y., Mu, J., Ding, C., Xue, J., Li, S., Zhou, S., Wang, A. and Yu, J.

Bioavailability Evaluation of a New Compound Bone Peptide Formula.

DOI: 10.5220/0011217300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 467-470

ISBN: 978-989-758-595-1

467

Bone peptide is rich in a variety of non-essential

amino acids, such as glycine (Gly), proline (Pro) and

hydroxyproline (Hyp). Hyp is the main component of

collagen tissue, and it is a specific amino acid in

collagen, accounting for about 13% of the total

collagen amino acid (SatoAsai 2020, Wang 2020,

Watanabe 2010). Hyp in blood is a degradation

product of various collagen peptides and is used as a

marker of bone absorption. By measuring the content

of hyp in the serum, the absorption and bioavailability

of bone peptide in mice after sample administration

can be measured (Yazaki 2017). In addition, previous

studies have shown that mice can directly absorb

collagen Gly-Pro-Hyp in the bone peptide sample

polypeptide mixture. Therefore, the measurement of

collagen peptides in mice is another key indicator to

evaluated bone peptide absorption and

bioavailability. In this study, the contents of Hyp and

Gly-Pro-Hyp in mouse plasma were measured after

administration for different times. The bioavailability

of various bone peptide samples was evaluated by

simulating the area model under the curve of drug

time, which provided basic research basis for the

development and application of new bovine bone

peptide products.

2 MATERIALS AND METHODS

2.1 Material

Bone peptide composite powder (Hangzhou

baibeiyou Biotechnology Co., Ltd.); Bone peptide

tablet (Jilin Huakang Pharmaceutical Co., Ltd.

National medicine quasi H20058927); Injection of

compound bone peptide (Nanjing Xinha

Pharmaceutical Co., Ltd. National medicine quasi

H20003533). All samples are stored at 4℃.

2.2 Determination of Serum Index

Preparations of serum: the blood was taken from the

aorta of mouse eyeball and placed in a centrifugal

tube with plug. After standing at room temperature

for 30 min, the blood was centrifuged at 4000 rpm for

10 min. The upper serum was slowly taken out by

pipette and placed in another centrifuge tube with

plug for determination of serum indexes.

2.3 Detection of Hyp in Serum

Healthy C57/BL mice were selected and divided into

6 groups: blank control group, positive

pharmaceutical control group 1 (bone peptide

injection group), positive pharmaceutical control

group 2 (bone peptide group), low (1.6 g/kg), medium

(3.2 g/kg) and high (4.8 g/kg) dose of bone peptide.

After adaptive for a week, 20 mice in each group were

gavaged once. 4 mice were sacrificed at 1, 3, 6, 12,

24 h in in each group and the plasma of each mouse

was obtained. Then the content of hyp in the serum

was determined, and the bioavailability of the

samples was analyzed. The AUC of Hyp was

calculated to compare the effective exposure amount

of drug absorption.

2.4 Detection of Gly-Pro-Hyp

The mice of positive pharmaceutical control groups

and the experimental groups were gavaged and

sacrificed the same as above. The content of Gly-Pro-

Hyp in the serum was determined, and the

bioavailability of the samples was analyzed. The

AUC of Gly-Pro-Hyp was calculated to compare the

effective exposure amount of drug absorption.

3 RESULTS AND ANALYSIS

3.1 Determination of Hyp Content in

the Serum of Mice

Figure 1: The content of Hyp in serum A: Blank group; B:

Low dose group (1.6 g/kg); C: Medium dose group (3.2

g/kg); D: High dose group (4.8 g/kg); E: Bone peptide tablet

group (576 mg/kg); F: Injection group (90 mg/kg).

The content of Hyp in the serum of the experimental

group can be obtained in Figure 1. The level of Hyp

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

468

was increased in a dose-dependent manner in the

serum. The content of Hyp in the serum of bone

peptide samples was reached the highest level after 3

h, and the Hyp level remained high with the extension

of time, and no significant decrease of Hyp occurred

after 24 h. It indicated that the utilization rate of

compound bone peptide samples was significantly

higher than that of bone peptide tablet and bone

peptide injection.

3.2 Calculation of AUC of Serum Hyp

in Mice

(**** P <0.0001)

Figure 2: The AUC of Hyp in the serum of mice.

The AUC is the area under the curve obtained by

taking the blood concentration after administration as

the ordinate and time as the abscendant. In this study,

the integral method is used to obtain AUC, which

indicate that the drug absorbs in a certain amount of

time after taking a certain dose drug and is

proportional to the amount of drug absorbed by the

organism.

As shown in Figure 2, the AUC of Hyp was

increased in a dose-dependent manner. And the AUC

of low, medium, high-dose bone peptide samples of

Hyp is much higher than the clinical dose bone

peptide injection group and the bone peptide tablet

group. It proved that the advantage of effective

absorption of bone peptide samples. The results

showed that the effective exposure of clinical dosage

- low dose groups was significantly higher than that

of the bone peptide injection and bone peptide tablet.

3.3 Determination of Gly-Pro-Hyp

Content in the Serum of Mice

Figure 3: The content of Gly-Pro-Hyp in serum A: Blank

group; B: Low dose group (1.6 g/kg); C: Medium dose

group (3.2 g/kg); D: High dose group (4.8 g/kg); E: Bone

peptide tablet group (576 mg/kg); F: Injection group (90

mg/kg)

There is a significant increase in the content of Gly-

Pro-Hyp in each experimental group mouse serum.

The level of Gly-Pro-Hyp was increases in a dose-

dependent manner in the serum. And the level of Gly-

Pro-Hyp content is reached the highest after 3 h. The

Gly-Pro-Hyp content was remained high, and no

significant decrease of Gly-Pro-Hyp occurred after

24 h. It indicated that the utilization rate of the bone

peptide samples is significantly higher than the bone

peptide tablet and bone peptide injection.

3.4 Calculation of AUC of Serum

Gly-Pro-Hyp in Mice

(****P<0.0001)

Figure 4: The AUC of Gly-Pro-Hyp in the serum of mice.

Bioavailability Evaluation of a New Compound Bone Peptide Formula

469

Using the same principle of Figure 2, the AUC of

Gly-Pro-Hyp was obtained using the integration

method. As shown in Figure 4, the AUC of Gly-Pro-

Hyp of bone peptide samples increased in a dose-

dependent manner. And the AUC of low, medium

and high-dose bone peptide samples was higher than

the clinical dose bone peptide injection group and

bone peptide tablet group, which proved the

advantage of effective absorption of bone peptide

samples. It indicates that the effective exposure of

clinical dosage - low dose groups was significantly

higher than that of the bone peptide injection and

bone peptide tablet.

4 CONCLUSIONS

In summarize, the results showed that compared with

the blank negative control group, the levels of Hyp

and Gly-Pro-Hyp in the serum of the experimental

groups (positive medicinal bone peptide tablet,

positive medicinal bone peptide injection, low-dose

bone peptide samples and high-dose bone peptide

samples) were significantly increased.

The levels of Hyp and collagen Gly-Pro-Hyp in

mouse serum can reach the highest after at gavaged

for 3 h of the new bone peptide formulation sample,

and the levels of Hyp and collagen Gly-Pro-Hyp was

still remained at high level in 24 h. It indicates that

the utilization rate of mouse bone peptide samples is

significantly higher than that of bone peptide tablet

and bone peptide injection, and the action time of

bone peptide samples is significantly longer than that

of bone peptide tablet and bone peptide injection.

The AUC was obtained by simulating the

pharmacokinetic mathematical model of Hyp and

Gly-Pro-Hyp using the integration method. The

results revealed that the AUC of the new bone peptide

formulation samples increased in a dose-dependent

manner, indicating that the effective exposure of the

mice absorbing the bone peptide samples increased.

And the AUC of Hyp and Gly-Pro-Hyp of low-dose

bone peptide sample group was significantly higher

than those in the clinical dose bone peptide injection

and bone peptide tablet groups. It indicates that the

drug exposure of clinical use dose - low dose bone

peptide samples was significantly higher than that of

bone peptide injection and bone peptide tablet.

REFERENCES

Ahn, C. & J. Je (2019) Bone health-promoting bioactive

peptides. J.food biochemistry, 43, e12529.

Bello, A. E. & S. Oesser (2006) Collagen hydrolysate for

the treatment of osteoarthritis and other joint disorders:

a review of the literature. J.Current medical research

and opinion, 22, 2221-32.

Cao, S., Y. Wang, Y. Hao, W. Zhang & G. Zhou (2020)

Antihypertensive Effects in Vitro and in Vivo of Novel

Angiotensin-Converting Enzyme Inhibitory Peptides

from Bovine Bone Gelatin Hydrolysate. J. Agricultural

and Food Chemistry, 68, 759-768.

Lee, H., H. Jang, D. Ahn, H. Kim, H. Y. Jeon, D. B. Seo, J.

Lee, J. K. Choi & S. Kang (2019) Orally administered

collagen peptide protects against UVB-induced skin

aging through the absorption of dipeptide forms, Gly-

Pro and Pro-Hyp. J. Bioscience, biotechnology, and

biochemistry, 83, 1146-1156.

Li, P. & G. Wu (2018) Roles of dietary glycine, proline, and

hydroxyproline in collagen synthesis and animal

growth. J.Amino acids, 50, 29-38.

Pountos, I., M. Panteli, A. Lampropoulos, E. Jones, G. M.

Calori & P. V. Giannoudis (2016) The role of peptides

in bone healing and regeneration: a systematic review.

J.BMC medicine, 14, 103.

Rosano, C. L., C. B. Braun & C. Hurwitz. 1987. A method

for serum C1q based on its hydroxyproline content.,

398-400..

Sato, K., T. T. Asai & S. Jimi (2020) Collagen-Derived Di-

Peptide, Prolylhydroxyproline (Pro-Hyp): A New Low

Molecular Weight Growth-Initiating Factor for

Specific Fibroblasts Associated With Wound Healing.

J.cell and developmental biology, 8, 548975.

Wang, J., J. Liu & Y. Guo (2020) Cell Growth Stimulation,

Cell Cycle Alternation, and Anti-Apoptosis Effects of

Bovine Bone Collagen Hydrolysates Derived Peptides

on MC3T3-E1 Cells Ex Vivo. J.Molecules, 25.

Watanabe-Kamiyama, M., M. Shimizu, S. Kamiyama, Y.

Taguchi, H. Sone, F. Morimatsu, H. Shirakawa, Y.

Furukawa & M. Komai (2010) Absorption and

effectiveness of orally administered low molecular

weight collagen hydrolysate in rats. J.agricultural and

food chemistry, 58, 835-41.

Yazaki, M., Y. Ito, M. Yamada, S. Goulas, S. Teramoto, M.

Nakaya, S. Ohno & K. Yamaguchi (2017) Oral

Ingestion of Collagen Hydrolysate Leads to the

Transportation of Highly Concentrated Gly-Pro-Hyp

and Its Hydrolyzed Form of Pro-Hyp into the

Bloodstream and Skin. J.agricultural and food

chemistry, 65, 2315-2322.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

470