CN116196337A

CN116196337A - Intravenous administration preparation for resisting aging

Info

Publication number: CN116196337A
Application number: CN202111453709.3A
Authority: CN
Inventors: 钱进
Original assignee: Zhejiang Chuyuan Biotechnology Co ltd
Current assignee: Zhejiang Chuyuan Biotechnology Co ltd
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2023-06-02

Abstract

The present invention provides intravenous formulations for anti-aging. In particular, the present invention relates to the use of amniotic fluid or a pharmaceutical composition comprising said amniotic fluid in the manufacture of an anti-ageing intravenous formulation, or in the manufacture of an intravenous formulation for reducing free radicals in a subject, or in the manufacture of an intravenous formulation for increasing superoxide dismutase and/or glutathione peroxidase levels in a subject; wherein the amniotic fluid is derived from an egg with an embryo age of 5-10 days, or from other poultry eggs except for a chicken with a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

Description

Intravenous administration preparation for resisting aging

Technical Field

The present invention relates to intravenous formulations for anti-aging.

Background

Aging is the process of loss and deterioration of the body from constituent materials, tissue structures to physiological functions. Physiological changes in the aging process of the human body are mainly reflected in the loss of tissue cells and constituent substances of the body, the slowing of the metabolic rate of the body and the hypofunction of the body and organs.

Scientific studies have shown that aging is associated with the production of excessive free radicals. The radical theory of aging suggests that degenerative changes in the aging process are due to deleterious effects of free radicals generated during normal cellular metabolism. Some in vivo and in vitro experiments show that free radical inhibitors and antioxidants can prolong the life of cells and animals; the ability to defend against free radicals in the body diminishes with age; vertebrates have long lives and low yields of oxygen radicals in vivo. Thus, by inhibiting the formation of free radicals in the body, aging can be prevented or anti-aging can be achieved.

Disclosure of Invention

The first aspect of the invention provides the use of amniotic fluid or a pharmaceutical composition containing said amniotic fluid in the preparation of an anti-ageing intravenous formulation; wherein the amniotic fluid is derived from an egg with an embryo age of 5-10 days, or from other poultry eggs except for a chicken with a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

In a second aspect the invention provides the use of amniotic fluid or a pharmaceutical composition comprising said amniotic fluid in the manufacture of a intravenous formulation for reducing free radicals in a subject, or for increasing superoxide dismutase and/or glutathione peroxidase levels in a subject; wherein the amniotic fluid is derived from an egg with an embryo age of 5-10 days, or from other poultry eggs except for a chicken with a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

In a third aspect the present invention provides the use of amniotic fluid or a pharmaceutical composition comprising said amniotic fluid in the manufacture of a intravenous formulation for increasing superoxide dismutase and/or glutathione peroxidase levels in a subject, thereby reducing free radicals in the subject, anti-aging; wherein the amniotic fluid is derived from an egg with an embryo age of 5-10 days, or from other poultry eggs except for a chicken with a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

In one or more embodiments, the amniotic fluid is derived from an egg having an embryo age of 6-9 days or a duck egg having an embryo age of 8-9 days, preferably an egg having an embryo age of 7-8 days, more preferably an egg having an embryo age of about 7 days.

In one or more embodiments, the intravenous administration preparation contains the amniotic fluid and physiological saline for injection, water for injection and/or glucose injection.

In one or more embodiments, the intravenous formulation is an intravenous injection or an intravenous infusion.

In one or more embodiments, the intravenous formulation contains 5-100% (v/v) or 10-35% (v/v) amniotic fluid, preferably 50-100% (v/v) amniotic fluid.

In one or more embodiments, the intravenous formulation is administered multiple times per day, twice per day, every two days, every three days, every four days, every five days, or every six days, every half month, or once a month, several months, or once a year.

In one or more embodiments, the intravenous formulation is a lyophilized formulation.

Drawings

Fig. 1: anti-free radical ability of amniotic fluid (EE) of chicken eggs of different embryo ages. Wherein the abscissa represents embryo age and the ordinate represents clearance.

Fig. 2: effect of amniotic fluid from chicken eggs on the growth viability and migration ability of Human Umbilical Vein Endothelial Cells (HUVECs). Wherein the abscissa represents the culture medium and the ordinate represents the OD ₄₅₀ Values.

Fig. 3: amniotic fluid derived from duck eggs for chicken embryo fibroblastsInfluence of growth vigor and migration ability. Wherein the abscissa represents the culture medium and the ordinate represents the OD ₄₅₀ Values.

Fig. 4: survival during injection to pre-anatomic D7 EE-1 group and placebo-1 group, n=7.

Fig. 5: mean body weight from injection period to pre-dissection D7 EE-1 and placebo-1, n=7.

Fig. 6: d7 EE-1 and placebo-1 group blood routine examination: a) white blood cell line count, B) red blood cell line count, C) platelet count, n=7.

Fig. 7: d7 EE-1 group and blank-1 group serum biochemical examination: a) Serum total superoxide dismutase (SOD), B) serum total glutathione peroxidase (GSH-PX), n=7.

Fig. 8: the D7 EE-1 and placebo-1 mice were dissected, weighted for Heart (Heart), liver (Liver), spleen (Spleen), lung (Lung), kidney (Kidney), pancreas (pancrees), brain (Brain) and organ weight ratios were calculated.

Fig. 9: average body weight of D8 EE, D9 EE, D10 EE and placebo group (a) and survival (B).

Fig. 10: average body weights of D8 EE, D9 EE, D10 EE and placebo groups were injected.

Fig. 11: d11EE and D14 EE were significantly biotoxic to mice.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

The inventors found that intravenous formulations of amniotic fluid of non-human animals with embryo ages of 11 days and 14 days were significantly biotoxic to subjects when given intravenous injection, and exhibited increased mortality due to acute immune response after injection. In contrast, intravenous injection of amniotic fluid of a non-human animal having an embryo age of 5 to 10 days was not observed to have a significant effect on the survival rate of the subject when intravenous injection was performed. Meanwhile, the aging of the subject can be significantly delayed by intravenous injection, thereby completing the present invention.

Accordingly, the present invention provides an intravenous administration preparation comprising amniotic fluid; wherein the amniotic fluid is derived from an egg having an embryo age of 5-10 days, preferably an egg having an embryo age of 6-8 days, more preferably an egg having an embryo age of about 7 days, or from an egg of a bird other than a chicken having a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

In the present invention, amniotic fluid may be derived from avian eggs and non-human mammals. Poultry eggs refer to poultry eggs. Preferred birds are poultry such as chickens, ducks and geese. Preferably, the present invention uses eggs that are embryonated for 5-10 days, preferably 6-9 days. It is understood that the proper embryo age may not be the same for different eggs. For example, when using eggs, it is preferable to use eggs having an embryo age of 5 to 10 days, more preferably 6 to 9 days, even more preferably 7 to 8 days, and even more preferably about 7 days. When eggs of other birds are used, eggs whose development period corresponds to that of the embryonated chicken eggs described above may be used. For example, when using duck eggs, duck eggs having an embryo age of 8-10 days, especially 8-9 days, may be best.

The egg amniotic fluid can be obtained by a conventional method. For example, the blunt end of an egg of the corresponding embryo age may be knocked to fracture the eggshell and the eggshell peeled apart to form a mouth having a diameter of about 2 cm. The shell membrane and vitelline membrane were then carefully torn apart with forceps, taking care not to disrupt the amniotic membrane. The amniotic membrane and the connective tissue which are wrapped with the embryo are poured into a culture dish from the shell, and the amniotic membrane is penetrated by a syringe to extract amniotic fluid until the amniotic membrane clings to the embryo, thereby obtaining the amniotic fluid used in the invention.

The amniotic fluid may also be derived herein from a non-human mammal, in particular a rodent, such as from a mouse. Other non-human mammals may be common domestic animals such as cattle, sheep, dogs, cats, pigs, etc. In certain embodiments, the amniotic fluid is from an embryo of a rodent that is 8-12 days old, or from a non-human mammal whose developmental stage corresponds to the developmental stage in which the rodent is 8-12 days old. Amniotic fluid can be obtained by conventional methods. For example, the amniotic fluid used in the present invention can be obtained by cutting the abdominal cavity of a pregnant mouse for 8-12 days with surgical scissors, carefully taking out and cutting the uterus, and puncturing the amniotic membrane with a syringe to extract the amniotic fluid until the amniotic membrane clings to the embryo.

It will be appreciated that the amniotic fluid may be centrifuged if necessary to separate out impurities which may be present, such as yolk, etc., to obtain as pure an amniotic fluid as possible. The supernatant obtained after centrifugation is the amniotic fluid used in the invention. It should be understood that all steps for obtaining amniotic fluid are performed under sterile conditions; in addition, "amniotic fluid" as used herein shall mean "pure" amniotic fluid, i.e., amniotic fluid isolated from avian eggs or non-human mammalian embryos that does not contain other components within avian eggs or non-human mammalian embryos, nor is contaminated with foreign materials. Pure amniotic fluid can be stored in a refrigerator below-60 ℃ and used after thawing.

The amniotic fluid described herein may be used as an active ingredient of a medicament for in vivo administration to a subject in need thereof. For example, an effective amount of an amniotic fluid described herein, or a pharmaceutical composition containing the amniotic fluid, may be administered to a subject in need thereof.

Herein, the subject may be an animal, such as a mammal, in particular a human.

In a particularly preferred embodiment of the present invention, amniotic fluid, in particular, an avian egg amniotic fluid as described herein, more preferably an egg amniotic fluid, is used to prepare an intravenous formulation for administration to a subject in need thereof for anti-aging.

Aging, as used herein, refers to the phenomenon of progressive decline in the physical and psychological adaptive capacity of the body to the environment and the gradual tendency to die. The radical theory of aging suggests that degenerative changes in the aging process are due to deleterious effects of free radicals produced during normal cellular metabolism. Free radicals are intermediate products of normal metabolism, have strong reaction capacity, can oxidize various substances in cells and damage biological membranes. And can crosslink macromolecules such as proteins, nucleic acids and the like to influence the normal functions of the macromolecules. Some in vivo and in vitro experiments show that free radical inhibitors and antioxidants can extend the life of cells and animals; the ability to defend against free radicals in the body diminishes with age; vertebrates have long lives and low yields of oxygen radicals in vivo. The preparation can obviously improve the serum level of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), thereby obviously reducing free radicals in animals and realizing the anti-aging effect.

Pharmaceutical compositions containing amniotic fluid described herein typically also contain pharmaceutically acceptable excipients. Herein, "pharmaceutically acceptable excipients" refers to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, including, but not limited to: antibiotics, humectants, pH adjusting agents, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavoring agents, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, or other suitable inactive ingredients for use with the pharmaceutically effective compounds. In some embodiments, the pharmaceutical composition is a pharmaceutical formulation suitable for intravenous administration, which contains a pharmaceutically acceptable adjuvant suitable for intravenous administration.

In some embodiments, a therapeutically effective amount of amniotic fluid may be mixed with an appropriate amount of physiological saline for injection, water for injection or glucose injection, and then administered for anti-aging by, for example, intravenous injection or intravenous infusion. In some embodiments, the intravenous formulation may contain 5-100% (v/v) or 10-100% amniotic fluid described herein, preferably 50-100%. In some embodiments, the intravenous administration preparation can be in a freeze-dried preparation form, and can be dissolved by pharmaceutically acceptable auxiliary materials such as physiological saline for injection, water for injection, glucose injection and the like to prepare intravenous administration preparation such as injection or infusion.

In this case, the dosage and frequency of administration may be determined by one skilled in the art according to the specific condition, the age and sex of the subject to be administered, and the like. Administration may be daily or weekly injections. In certain embodiments, the frequency of administration may be multiple times per day, twice per day, every two days, every three days, every four days, every five days, or every six days, every half month, or once a month, several months, or once a year. Typically, the individual will be administered a dose in the range of 0.1mL/kg body weight to 2mL/kg body weight.

In some embodiments, the invention provides the use of an amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid in the preparation of an intravenous formulation for anti-aging. In still other embodiments, the invention provides the use of an amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid in the preparation of an intravenous formulation for reducing free radicals in a subject. In still other embodiments, the invention provides the use of amniotic fluid described herein or a pharmaceutical composition containing the same, in the preparation of an intravenous formulation for increasing the level of superoxide dismutase (SOD) and/or glutathione peroxidase (GSH-PX) in a subject. In some embodiments, the invention provides the use of an amniotic fluid described herein or a pharmaceutical composition containing the amniotic fluid in the preparation of a intravenous formulation for increasing superoxide dismutase (SOD) and/or glutathione peroxidase (GSH-PX) levels in a subject, reducing free radicals in a subject, thereby preventing aging. In some embodiments, the intravenous formulation contains the amniotic fluid and physiological saline for injection, water for injection, and/or glucose injection. In some embodiments, the intravenous formulation is an intravenous injection or an intravenous infusion.

The invention also provides a method of anti-aging comprising intravenously administering to a subject in need thereof an amniotic fluid as described herein, a pharmaceutical composition comprising the amniotic fluid, or an intravenous formulation as described herein.

In particularly preferred embodiments of the pharmaceutical compositions, intravenous formulations, methods and uses of the invention, the amniotic fluid is preferably from an egg having an embryo age of about 7 days.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods, reagents and apparatus used in the examples are those conventional in the art unless otherwise indicated.

Example 1: acquisition of amniotic fluid (EE) of eggs with different embryo ages

1. Material

a) Instrument and tool

Microcomputer full-automatic incubator (Zhengda) ^TM ZF 880), clean petri dishes, 1.0mL syringe (Jiangxi Hong Da) ^TM ) Forceps sterilized by 70% alcohol, stainless steel screen, sterile centrifuge tube

# SCT-50 ML-25-S) and a low-speed cryocentrifuge (well-appreciated KDC-2046).

b) Reagents and biological materials

Eggs of different embryogenic ages.

2. Experimental procedure

Taking eggs, knocking the flat blunt end which is placed upwards to fracture the eggshells, stripping the eggshells to form a mouth with the diameter of about 2 cm, and flattening the edges as much as possible. The shell membrane and vitelline membrane were carefully torn with forceps, taking care not to destroy the amniotic membrane. And observing the embryo development condition, and only embryos which are well developed and meet the corresponding stage standard can be used for extracting amniotic fluid.

Pouring the amniotic membrane and the connective tissue which are wrapped with the embryo from the shell into a culture dish, penetrating the amniotic membrane by using a syringe to extract amniotic fluid, enabling the inclined surface of the needle opening to face away from the embryo until the amniotic membrane clings to the embryo, and then injecting the clarified, colorless and foreign-body-free amniotic fluid into a centrifuge tube in an ice box.

Taking out embryo in amniotic membrane with forceps, collecting in stainless steel sieve placed on ice, homogenizing the collected embryo every one hour with stirrer, packaging in sterile plastic storage tank, and placing in refrigerator at-80deg.C. Can be vertically placed after freezing.

Through the Meinaida ^TM 1800 ultraviolet light splittingThe collected amniotic fluid extract is tested by a photometer, and standard operation procedures of the photometer are referred to a manual.

Balancing a centrifuge tube for collecting amniotic fluid extract, and then using Zhongjia ^TM KDC-2046 low-speed cryocentrifuge was centrifuged at 3500rpm for 20min at 5 ℃ (for standard protocols for centrifuge operation see instruction manual). The supernatant was decanted and transferred to a clean plastic storage tank and stored in a-80 ℃ refrigerator. 5mL of the swatches were reserved for subsequent testing per batch.

All steps are performed under aseptic conditions.

Example 2: test of EE anti-radical Capacity

DPPH is a 1, 1-diphenyl-2-picrylhydrazyl radical having the structure shown below:

in DPPH molecule, due to the presence of multiple electron-withdrawing-NO ₂ And a large pi bond of the benzene ring, so that the nitrogen radical can exist stably.

As DPPH radicals are scavenged, the absorbance A at 519nm of their absorption maxima decreases. The stable free radical DPPH provides an ideal and simple pharmacological model for detecting the activity of scavenging the free radical. This example uses DPPH to detect anti-radical capability from EE.

Dissolving 0.8mg DPPH in 20mL methanol solvent, performing ultrasonic treatment for 5min, and shaking thoroughly to make the upper and lower parts uniform. 1mL of the DPPH solution was taken and A was measured at 519nm ₀ Value, a=0.5-0.7. The DPPH solution is preserved in dark and used up within 3.5 hours.

The amniotic fluid of eggs with embryo ages of 6 days, 7 days, 8 days, 9 days, 10 days and 11 days is obtained by the method in the first embodiment, and is stored in a refrigerator at 4 ℃ for standby after centrifugation.

Standard curves were determined using vitamin C as a positive control. Taking Vc samples with different volumes and 0.04mg/mL, adding DPPH with 0.6mL, adding absolute ethyl alcohol to complement to 1mL, uniformly mixing, taking methanol as a control for zeroing, and measuring the light absorption value under 519nm wavelength. The data were repeated three times and plotted.

Adding 400 mu L of amniotic fluid with different embryo ages into a test tube, adding 600 mu L of the prepared methanol solution of DPPH, uniformly mixing, and reacting for 10min to ensure that bubbles are not generated (uniform mixing is performed before measurement). The absorbance at 519nm was measured by zeroing with methanol as a control.

The loading information for each group is shown in table 1 below:

table 1: each component of

Experimental group	Sample liquid	95% ethanol (or absolute ethanol)	DPPH test solution	Total volume of
					Blank group	0mL	0.4mL	0.6mL	1mL
Vc	nμL	(400-n)μL	0.6mL	1mL
					Sample group	0.4mL	0mL	0.6mL	1mL

Clearance (inhibition) was calculated using the following formula:

clearance (%) = (a) ₀ -A)/A ₀ ×100％。

The results are shown in FIG. 1.

Example 3: scratch test of egg amniotic fluid and duck egg amniotic fluid

Amniotic fluid of duck eggs with embryo age of 8 days was obtained by the same method as in example 1. Scratch experiments were used to test the effect of chicken egg amniotic fluid on Human Umbilical Vein Endothelial Cells (HUVEC) and duck egg amniotic fluid on the growth viability and migration ability of chicken embryo fibroblasts. Human umbilical vein endothelial cells are obtained from commercial sources.

The composition of the DMEM medium used in this example was as follows:

#Cat.11960077, 1% L-glutamine (++>

# G0200) and 5% FBS (++>

#Cat.10099141); other reagents: 0.25% pancreatin (Hangzhou family easy organism) ^TM #CY003)，PBS(BI ^TM #02-024-1 ACS), 0.4% trypan blue stain (BBI) ^TM #72-57-1)。

Preparing a 6-hole plate on the first day before the experiment, and drawing 5-6 uniformly distributed transverse lines on the back of the 6-hole plate by using a mark pen and using a ruler to traverse the through holes; and drawing a vertical line at the center line to indicate the position of the scratch. About 5X 10 was added to each well ⁵ Cells in the logarithmic growth phase, in principle, reached a fusion rate of 90% after overnight inoculation.

On the day of the experiment, the bottom surface of the 6-well plate is marked with a gun head which is vertical to the bottom surface of the 6-well plate along the vertical line of the mark pen compared with a straight ruler. The gun head is not inclined and bent as much as possible, and the same gun head is preferably used among different holes, and the width is preferably 1000-2000 um. Each well was washed 3 times with 2mL PBS and cells at the scratch were washed away. 2mL of culture medium containing EE with different contents is added into each hole respectively, and the culture is carried out conventionally, and liquid is changed every 48 hours. The time from scratch was 0h, every 24h was photographed at fixed points, and the cell spacing on both sides of the scratch was measured. Observing the growth of cells in each well; drawing a graph by taking time (days) as a horizontal axis and the scratch distance in each hole as a vertical axis; the rate of scratch healing in each well was calculated.

The results are shown in FIGS. 2 and 3. Figure 2 shows the effect of amniotic fluid from eggs on the growth viability and migration capacity of Human Umbilical Vein Endothelial Cells (HUVEC), the addition of 5% (volume ratio) amniotic fluid clearly has a very significant promoting effect on the healing of HUVEC. Figure 3 shows the effect of amniotic fluid from duck eggs on the growth vigor and migration ability of chicken embryo fibroblasts, and the addition of amniotic fluid also shows a very significant promoting effect on the healing of chicken embryo fibroblasts.

The results show that amniotic fluids from different sources all have the same or similar biological activity.

Example 4: short-term intravenous injection of chick embryo amniotic fluid on KM mice

This example tests the effect of short-term intravenous injection of EE on KM mice.

Experimental animals were male KM mice (vitelliwa, zhejiang) 6-8 weeks old, n=7 per group.

Amniotic fluids of different embryo ages were obtained by the method of example 1 and used in this example.

The experimental method comprises the following steps: tail vein injection, 0.15 mL/dose, 7 times in 10 days, and continuous observation state in the range of injection days. Weigh and observe during the experiment. After the experiment is finished, sampling and dissecting are performed, and organs such as gravity center, liver, spleen, lung, kidney, pancreas, brain and the like are observed and called.

The reference substance is 0.9% sodium chloride injection

100ml:0.9 g), lot number: lot#2007061305.

The experimental set-up is shown in table 2 below:

TABLE 2

Group of	Operation of
		D7 EE-1	7 injections of D7 EE 0.15mL EE per dose were made over 10 days
Blank-1	7 times of 0.9% sodium chloride injection solution are injected within 10 days, and each time is 0.15 mL/dose

The experimental procedure is shown in table 3 below:

TABLE 3 Table 3

Days (days)	Tail vein injection	Remarks
				0	①0.15mL	/
2	②0.15mL	/
			3	③0.15mL	/
6	④0.15mL	/
			7	⑤0.15mL	/
9	⑥0.15mL	/
			10	⑦0.15mL	D7 EE-1&Blood sampling dissection on 13 th day of Blank-1

Note that: (1) - (7) means 1 st to 7 th injections, each injection being weighed before; the injection time of the Blank-1 group is the same as that of the D7 EE-1 group; d7 EE refers to amniotic fluid obtained from eggs with an embryo age of 7 days.

Sampling and detection are shown in table 4 below:

TABLE 4 Table 4

Results:

1. survival rate

Survival rates of D7 EE-1 and Blank-1 groups (Blank-1) were 100% and the survival curves were coincident within 10 days and 7 times before tail vein injection to dissection. The results are shown in FIG. 4.

2. State observation

The weight results of the animals in each experimental group are shown in fig. 5. D7 The initial values of the average body weights of the EE-1 group and the blank-1 group were 44.4g, and after 7 times of tail vein injection within 10 days, the average body weights of the D7 EE-1 group and the blank-1 group were 45.7g and 46.6g, respectively, and the growth rates were 2.86% and 4.99% respectively, and there was no significant difference between the groups (FIG. 5).

3. Blood routine

The results of the blood routine examination are shown in FIG. 6. Three lines of blood cells of the comparative D7 EE-1 group and the blank-1 group: white blood cell lines (FIG. 6, A), red blood cell lines (FIG. 6, B) and platelets (FIG. 6, C). D7 EE-1 group white blood cell count average was 0.36×10 ⁹ Per L, significantly lower than the mean value of the white blood cell count of the blank-1 group by 5.96×10 ⁹ L (reference range 0.8-6.8X10) ⁹ /L)。

Wherein the ratio of each type of white blood cell count and the reference value are (D7 EE-1 group/blank-1 group, reference range): neutrophil count (0.10/0.26,0.1-1.8), lymphocyte count (0.20/5.29,0.7-5.7), and monocyte count (0.06/0.31,0-0.3). D7 The EE-1 group of leukocytes was significantly below normal, mainly as demonstrated by lymphocyte concentrations below normal. Both erythrocyte lines and platelet counts were in the normal range with no significant differences between the groups.

4. Anti-aging capability (SOD, GSH-PX)

Serum was biochemically examined for superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). The results are shown in FIG. 7.

As key peroxidation resistance indexes of organisms, the levels of SOD and GSH-PX can reflect the anti-aging capability of the organisms and the aging degree thereof [ Yang, G.Y. and the like, 2013,Effects of cigarette smoke extracts on the growth and senescence of skin fibroblasts in vitro,International journal of biological sciences,9 (6), 613-623; wang, F. Et al, 2016, seniscence-specific change in ROS scavenging enzyme activities and regulation of various SOD isozymes to ROS levels in psf mutant rice leaves, plant physiology and biochemistry: PPB,109, 248-261]. As can be seen from fig. 7, the serum SOD and GSH-PX levels of the D7 EE-1 group were higher than those of the blank-1 group, and the GSH-PX levels were significantly different, indicating that the D7 EE-1 group mice had a stronger anti-aging capacity.

5. Anatomic and pathological observations

After blood collection, mice were sacrificed by cervical dislocation and the following organs were dissected: heart, liver, spleen, lung, kidney, pancreas, brain. The morphology of each organ was observed in the D7 EE-1 group and the blank-1 group, and no significant abnormality was found.

Each organ is weighed and the organ weight is calculated as shown in fig. 8. D7 The body weight ratio of each vital organ of EE-1 group and Blank-1 group mice is at normal level, and there is no obvious difference between groups.

Example 5: effect of medium-term intravenous injection of chick embryo amniotic fluid on KM mice

This example tests the effect of medium-term intravenous injection of EE on KM mice.

Experimental animals were male KM mice (vitelliwa, zhejiang) 6-8 weeks old, n=6 per group.

Amniotic fluids (D8 EE, D9 EE and D10 EE) were obtained for 8 days, 9 days and 10 days of embryo age using the procedure of example 1 for this example.

The experimental method comprises the following steps: tail vein injections, 0.15 mL/dose, were performed as in table 3 for the first 7 injections over 151 days, followed by 1 injection per week for a total of 26 injections. The status was continuously observed over the number of days of injection. Weigh and observe during the experiment.

The reference substance is 0.9% sodium chloride injection

100mL:0.9 g), lot number: lot#2007061305.

The experimental results are shown in FIG. 9. D8 EE and D9 EE were observed to promote weight gain in KM mice (FIG. 9, A), and no significant effect of D8 EE, D9 EE and D10 EE on KM mice survival was observed (FIG. 9, B; note: 1 case of death in the initial experimental period except for the D8 EE group, survival rates of each group were 100%).

Example 6: influence of Long-term intravenous injection of chick embryo amniotic fluid on KM mice

This example tests the effect of long-term intravenous injection of EE on KM mice.

The experimental method comprises the following steps: tail vein injection, 0.15 mL/dose, 7 times in 48 days, then 1 time per week, and the status was observed continuously over the number of days of injection. Weigh and observe during the experiment.

The reference substance is 0.9% sodium chloride injection

100mL:0.9 g), lot number: lot#2007061305.

The experimental results are shown in FIG. 10. The weight gain of KM mice in the long-term injection group D7 EE was observed to be slower than that of the groups D8 and D9 EE, and the hair color was glossy compared with the other groups.

Example 7: d11EE and D14 EE have significant biotoxicity to mice

Amniotic fluids (D7 EE, D11EE and D14 EE) of 7 days, 11 days and 17 days of embryo age were obtained for this example using the procedure of example 1.

The experimental method comprises the following steps: tail vein injection, 0.15 mL/dose, injection more than 40 times. Weigh and observe during the experiment.

The reference substance is 0.9% sodium chloride injection

100mL:0.9 g), lot number: lot#2007061305.

The experimental results are shown in FIG. 11. D11EE and D14 EE were significantly biotoxic in KM mice by 10 intravenous injections within 30 days, mainly manifested by increased mortality due to acute immune response following injection. The survival rate of the D11EE and D14 EE groups is 0 in a longer period (more than 40 days).

Claims

1. Application of amniotic fluid or a pharmaceutical composition containing the amniotic fluid in preparing an anti-aging intravenous administration preparation;

wherein the amniotic fluid is derived from an egg with an embryo age of 5-10 days, or from other poultry eggs except for a chicken with a development period corresponding to the development period in which the embryo-age egg is located; or from an embryo of a rodent with a gestational age of 8-12 days, or from a non-human mammal other than a rodent with a developmental stage corresponding to the developmental stage of a rodent with a gestational age of 8-12 days.

2. Use of amniotic fluid or a pharmaceutical composition comprising said amniotic fluid in the manufacture of a intravenous formulation for reducing free radicals in a subject, or in the manufacture of a intravenous formulation for increasing superoxide dismutase and/or glutathione peroxidase levels in a subject;

3. Use of amniotic fluid or a pharmaceutical composition comprising said amniotic fluid in the manufacture of a intravenous formulation for increasing superoxide dismutase and/or glutathione peroxidase levels in a subject, thereby reducing free radicals in the subject, and combating aging;

4. Use according to any one of claims 1 to 3, wherein the amniotic fluid is derived from an egg with an embryo age of 6 to 9 days or a duck egg with an embryo age of 8 to 9 days.

5. Use according to claim 4, wherein the amniotic fluid is derived from an egg having an embryo age of 7-8 days, preferably an egg having an embryo age of about 7 days.

6. The use according to any one of claims 1 to 5, wherein the intravenous formulation contains the amniotic fluid and physiological saline for injection, water for injection and/or glucose injection.

7. The use according to any one of claims 1 to 6, wherein the intravenous formulation is an intravenous injection or an intravenous infusion.

8. The use according to any one of claims 1 to 7, wherein the intravenous formulation contains 5-100% (v/v) amniotic fluid.

9. The use according to claim 8, wherein the intravenous formulation more preferably comprises 50-100% (v/v) amniotic fluid.

10. The use according to any one of claims 1 to 9, wherein the intravenous formulation is a lyophilized formulation.