# Patent

## Title
Compounds and method for inhibiting the activity of gelatinase and collagenase

## Abstract

Compounds extracted from Rhodiola rosea have ability to inhibit gelatinases and collagenases activity. The compounds have the chemical structure as shown below.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the priority benefit of Taiwan application serial no. 099123862, filed Jul. 20, 2010, the full disclosure of which is herein incorporated by reference.
BACKGROUND
1. Technical Field
The disclosure relates to compounds and method for inhibiting the activity of gelatinase and collagenase.
2. Description of Related Art
The ageing of human skin can divide into internal and external factors in human body. Internal factors are intrinsic aging (chronological aging), which is mainly regulated by genes. External factors known as extrinsic aging or photoageing are mostly influenced by environmental stimulus (such as UV radiation, free radicals, smoking, staying up and etc). Although the mechanism in the clinical and pathological aspects of these two ageing are significant different, the increment of matrix metalloproteinase (MMPs) expression are both involved. MMPs are zinc dependent endopeptidases. MMPs are regulated by TIMPs (inhibitors of metalloproteinases), and the amount of MMPs presence equilibrium under normal condition. However, the expression of MMPs increases when expose to external stimulus, such as long-term exposure of UV irradiation, the degradation of extracellular matrix (ECM) will be performed as resulting in skin aging.
SUMMARY
Therefore, the invention herein provides compounds and a method for gelatinase and collagenase activity inhibition. The compounds with ability of gelatinase and collagenase activity inhibition are:







BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a one-dimensional 1H NMR spectrum of new compound 4GX.
FIG. 2 is a two-dimensional 1H-1H COSY spectrum of new compound 4GX.
FIG. 3 is a one-dimensional 13C spectrum of new compound 4GX.
FIG. 4 is a two-dimensional HMQC spectrum of new compound 4GX.
FIG. 5 is a two-dimensional HMBC spectrum of new compound 4GX.



DETAILED DESCRIPTION
In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.
Biological ageing is a process happened spontaneously over time, and however, it is complex in nature. The behavior of ageing can be divided into structural and functional recession. Rhodiola rosea is a kind of plant for anti-ageing, and thus the active skin anti-ageing ingredients in Rhodiola rosea must be examined.
As the mention above, matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases, which including collagenase and gelatinases. Collagen can be divided into 16 types, where collagen type IV is to maintain the normal delivery of skin nutrients and moisture, and also keep skin tightening for wrinkle prevention. When skin directly exposure under UV light or during aging process, the hydrolysis of collagen type IV by collagenase increase and the biosynthesis of collagen type IV decrease, as resulting in the skin structural collapse and wrinkling.
Gelatinase includes MMP-2 (called as gelatinase A) and MMP-9 (called as gelatinase B). Gelatinase is capable of degrading proteins like non-fibronectin, fibronectin and elastin in extracellular matrix (ECM). When the skin exposure under UV light, the secretion of MMP will increase, more proteins will be destroyed by MMP and resulting in skin ageing. Collagenase and gelatinase are applied in the following experiments to test the active ingredients of Rhodiola rosea. Collagenase is directly used in experiments. The gelatinase is applied based on gelatinase secreting cells in experiments.
I. Preparing and Screening of Rhodiola rosea 
Alcohol was applied to extract Rhodiola rosea roots to obtain the Rhodiola rosea alcohol extracts. The Rhodiola rosea alcohol extracts was first purified by partitioning with 1:1 volume of ethyl acetate and water, and then the water solution gained from the first purification was secondly purified again by butanol. After completing the previous purification processes, three parts of aqueous solutions (ethyl acetate solution, butanol solution and the water solution) were obtained from the Rhodiola rosea alcohol extracts.
In order to screen the desire compounds from three parts of aqueous solutions (ethyl acetate solution, the butanol solution and the water solution), screen of three aqueous solutions were taken placed, where screening method, Assay of Gelatinase and Assay of Collagenase, will be mentioned detail below. According to the results, the ethyl acetate solution was determined effective inhibition in the collagenase and gelatinase.
Afterwards, the ethyl acetate solution was purified and separated into 56 fractions by chromatography, and the fractions, effective inhibition in the collagenase and gelatinase, were selected for the further purification by IsoleraTM Flash Purification System chromatography. The compositions in the Rhodiola rosea extract are listed bellowed on Table 1, where 4GX is a new chemical compound. And the purification processes and the chromatography are presented in order on Table 2







TABLE 1







the purified chemical compounds, 4GX and 4G, from Rhodiola rosea.









Code




Name
Compound name
Chemical Structure





4GX
1,2,3,6-tetra-O-galloyl- 4-O-(4-hydroxybenzoyl)- β-D-glucopyranoside






 


4G
1,2,3,6-tetra-O-galloyl- β-D-glucose





























TABLE 2







the purification condition of 4GX by Isolera TM Flash


Purification System.








Chromatography
Mobile phase (volume ratio)





1. Normal phase chromatography
hexane:acetone = 1:4


2. Reverse phase chromatography
methanol:water = 2:1


3. Reverse phase high performance
acetonitrile:water = 1:2


chromatography









II. The Identification of 4GX Structure

Identification of the chemical structure of the new compound 4GX was conducted. First, negative-ion mode of high-resolution electron spray ionization mass spectrometer (HRESIMS) was conducted to detect the (M-H) molecular weight of 4GX, and the resulting molecular weight of 4GX is 907.1241 and molecular formula is C41H32O24.
One-dimensional proton NMR test (1H-NMR), carbon 13 NMR test (13C-NMR), and two-dimensional HMBC (Heteronuclear Multiple Bond Coherence), HMQC (Heteronuclear Multiple Quantum Coherence) and 1H-1H COSY (Proton-Proton Correlation Spectroscopy) tests were conducted to examine 4GX by 500 MHz NMR, where deuterium solvent used herein was CD3OD.
The spectrum of one-dimensional 1H NMR of 4GX is presented in FIG. 1, and there are 7 proton signals, δH 4.40 (1H, m), δH 4.41 (1H, m), δH 4.51 (1H, m), δH 5.58 (1H, dd, J=8.3, 9.6 Hz), δH 5.63 (1H, t, J=9.6 Hz), δH 5.92 (1H, t, J=9.6 Hz), and δH 6.24 (1H, d, J=8.3 Hz), in the spectrum.
The spectrum of two-dimensional 1H-1H COSY NMR of 4GX is presented in FIG. 2. In FIG. 2, it is observed that δH 6.24 and δH 5.58 are related, δH 5.58 and δH 5.92 are related, δH 5.92 and δH 5.63 are related, δH 5.63 and δH 4.40 are related, and δH 4.40, δH 4.41 and δH 4.51 are related. Therefore, it is assumed that previous signals are the proton signals of β-glucopyranosyl.
The spectrum of one-dimensional 13C NMR of 4GX is presented in FIG. 3, and the carbon peaks of 4GX can be observed. The spectrum of two-dimensional HMQC NMR of 4GX is presented in FIG. 4. It is observed that chemical shifts (δC) of 6 carbons (corresponding to chemical shift of the previous 7 protons) are 74.7, 63.7, 72.6, 70.5, 74.5 and 94.3, which match with the 6 carbons of β-glucopyranosyl respectively.
There are proton singlet signals δH 6.88, 6.95, 7.05 and 7.10 having integral value 2 respectively indicated from the one-dimensional 1H NMR spectrum of 4GX in FIG. 1. In addition, it is observed that the chemical shifts of two carbons are at the characteristics position of aromatic rings (δC 110.0-112.0) with reference to the two-dimensional HMQC NMR of 4GX in FIG. 4.
The spectrum of two-dimensional HMBC NMR of 4GX is presented in FIG. 5, there is the carbon-hydrogen interactions over a distance of 2-3 carbon bonds. In FIG. 5, long distance coupling between aromatic hydrogen signals, δH 6.88, 6.95, 7.05, 7.10 of 4GX (chemical shift signals of the corresponding carbons are δC 110.8, 110.9, 111.1, 110.8) and δC 166.7, 167.5, 167.8, 168.4 (—COO—) were taken placed. Therefore, it can be determined that 4GX contains 4 sets of galloyl groups.
Besides, it is observed that δC 166.7 and δH 6.24 (Glc-1) are related, δC 167.5 and δH 5.58 (Glc-2) are related, δC 167.8 and δH 5.92 (Glc-3) are related, δC 168.4 and δH 4.41 (Glc-6) are coupling in the HMBC spectrum in FIG. 5. Since part of β-glucopyranosyl structure of 4GX and 4 sets of galloyl group are related, it is assumed that 4 sets of galloyl groups are linked to Glc-1,Glc-2,Glc-3,Glc-6 of glucose respectively according to the description above.
Moreover, δH 6.75 (2H, d, J=8.7 Hz) and δH 7.78 (2H, d, J=8.7 Hz) are doublet signals, these coupling constants are all 8.7 Hz in 1H NMR spectrum in FIG. 1, and therefore these two sets of proton signals belong to the substituent structures of aromatic ring in para-position. Additionally, it is confirmed that 4GX contains part of benzoic acid structure, where proton signals and δC 167.1 (—COO—) are related indicated from HMQC spectrum in FIG. 4 and HMBC spectrum in FIG. 5. Since —COO— (δC 167.1) of benzoic acid relating to δH 5.63 (Glc-4) are observed, it is assumed that benzoic acid substituent, X, is linked to Glc-4.
Therefore, the structure of 4GX are determined as 1, 2, 3, 6-tetra-O-galloyl-4-O-(4-hydroxybenzoyl)-β-D-glucopyranoside. The spectrum of NMR mentioning above are all presented on Table 3.







TABLE 3







NMR of 4GX.














Hydrolysis of tannin
4-hydroxyl


Glucopyran
1H
13C
acyl group
benzoyl group


Glycosylation
(δH)
(δC)
(—COO—, δC)*
(—COO—, δC)*














C1
6.24
94.3
166.7



C2
5.58
72.6
167.5


C3
5.92
74.5
167.8


C4
5.63
70.5

167.1


C5
4.40
74.7


C6
4.41
63.7
168.4



4.51





*HMBC spectrum has cross peaks






III. Assay of Gelatinase

Incubation of HT-1080 Gelatinase Secreting Cell Line
HT-1080 cell line (ATCC:CCL-121) is a human skin fibroblasts tumor, which is a kind of adherent cell lines, secreting gelatinase, MMP-2 and MMP-9, to extracellular space.
The thawing process and primary cell culture was first accomplished. The vial containing HT-1080 cell line from liquid nitrogen was immersed immediately in water at 37° C. for one minute to thaw the cell line, then the cell line was transferred into a flask with bottom based area around 75 cm2, where the flask containing medium were 15 mL of RPMI-1640 (with phenol red) (the composition of RPM I-1640 is showed in Table 4), heat-inactivated fetal bovine serum (FBS) and 1% PSQ (2 mM of 100 U/mL penicillin, 100 μg/mL streptomycin and 100 μg/mL L-glutamine), followed by incubation under 5% CO2/air at 37° C. in 70% of humidity. 10% FBS was provided for cell nutrition and 1% PSQ was provided for the prevention of cross-contamination. Finally, the flask was put in the incubator.







TABLE 4







the composition of RPMI-1640 medium








Composition
Concentration (mg/L)










Amino acid








Glycine
10


L-Arginine
200


L-Asparagine
50


L-Aspartic acid
20


L-Cystine 2HCl
65


L-Glutamic Acid
20


L-Glutamine
300


L-Histidine
15


L-Hydroxyproline
20


L-Isoleucine
50


L-Leucine
50


L-Lysine hydrochloride
40


L-Methionine
15


L-Phenylalanine
15


L-Proline
20


L-Serine
30


L-Threonine
20


L-Tryptophan
5


L-Tyrosine disodium salt dihydrate
29


L-Valine
20







Vitamins








Biotin
0.2


Choline chloride
3


D-Calcium pantothenate
0.25


Folic Acid
1


Niacinamide
1


Para-Aminobenzoic Acid
1


Pyridoxine hydrochloride
1


Riboflavin
0.2


Thiamine hydrochloride
1


Vitamin B12
0.005


i-Inositol
35







Inorganic salts








Calcium nitrate (Ca(NO3)2•4H2O)
100


Magnesium Sulfate (MgSO4•7H2O)
48.84


Potassium Chloride (KCl)
400


Sodium Chloride (NaCl)
6000


Sodium Phosphate dibasic (Na2HPO4•7H2O)
800







Other Components








D-Glucose (Dextrose)
2000


Glutathione (reduced)
1


Phenol Red
5









When the cells were saturated in the flask medium, the next subculture step must be conducted. First of all, to adopt the cell line to growth in a suspension culture, the previous medium was removed out from the flask, the monolayer surface of the cells were washed with PBS solution (phosphate buffered saline), and then the cells were incubated with 1 mL of trypsin in the same flask for 5 minute at 37° C. Afterwards, 4 ml of RPMI-1640 (with phenol red) containing 1% PSQ and 10% FBS medium was applied to inactivate the trypsin activities in the flask.
After removing the old medium, the cell suspension solution was diluted with a fresh medium. The method of replacing the fresh medium was to transfer the cell medium from the flask to the centrifugal vials, the centrifugal process was conducted at 1200 rpm at 24° C., and followed by removing the supernatant from the vials. Afterwards, the medium (RPMI-1640 (with phenol red) containing 1% PSQ and 10% FBS) was added to dilute the cell suspension solution, where the volume of the medium was four times than cell suspension solution in the vial.
Finally, a subculture process was conducted by replacing the diluted cell suspension solution with a new flask containing 10-12 ml of the previous mentioned medium. After that, repeat the cell adoption and cell suspension steps routinely when the cells are saturate on the bottom of the flask at the time interval of 2 or 3 days.
The Pre-Treatment of the Cells
Before carrying out any experimental process of HT-1080 cell lines, in order to enable HT-1080 cells to secrete more MMP-2 and MMP-9 to extracellular space, the medium RPMI-1640 containing 0.5% FBS (without phenol red) was applied when conducting the subcultivation process. After centrifugation, the medium RPMI-1640 containing only 0.5% FBS was added into centrifugal vials to resuspend the cells, and 20 μL of cell suspension solution was transferred into eppendorfs to mixed with 20 μL Trypan blue for cell dyeing. Finally, calculation of the cell concentration was accomplished by using hemocytometer through optical microscope.
Suitable amount of the medium RPMI-1640 (based on the cell concentration obtained previous) containing only 0.5% FBS (without phenol red) was applied into the remaining the cell suspension solution, as the resulting concentration of the cell suspension solution around 5×105 cells/mL. Afterwards, 500 μL of cell suspension solutions was transferred onto 24-well cell culture plates, followed by 24 hours incubation at 37° C. for the further experiments.
MTT Cell Viability Assay
To test the cytotoxicity of 4GX and 4G to HT-1080 cell lines, the cell viability assay need to be conducted. MTT cell viability assay was chosen for this experiment.
The principle of a cell viability assay is the conversion of a yellow tetrazolium salt (MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) into a purple product formazan via lactate dehydrogenase in intact mitochondria, where formazan can be dissolved in dimethylsulfoxide (DMSO). Thus, the increasing yield of resulting product formazan stands for increasing the percentage of cell vitality.
The cell suspension solution (with concentration about 5×105 cells/mL) of the previous cell pre-treatment process was placed to a 24-well cell culture plates with 24 hours of incubation under 5% of CO2/air at 37° C. in 70% of humidity, in order to allow cells to grow adherent on the well. After 24 hours of incubation, 1 μL of the solutions containing chemical compounds, 4GX and 4G, prepared in different concentrations (5-100 μM) were applied into each well respectively as vehicle controls, whereas the well applied without any chemical compounds as blank. Finally, the 24-well cell culture plate was placed in incubator for 22 hours of incubation.
Subsequently, the following steps need to avoid the light. After 22 hours of incubation, 50 μL of 5.5 mg/mL MTT was applied in each well for 2 hours of incubation at 37° C. Afterwards, the supernatant was removed from 24-well cell culture plates, and 400 μL DMSO was applied to dissolve the purple product formazan for 15 minutes by shaker at 50 rpm. 150-200 μL of the solution were taken out from each well to examine the adsorption value by ELISA at wavelength 550 nm.
The formula for calculation of cell vitality is presented following:

(Adsorption value of vehicle control/blank)×100%

The results of the cell survival test are showed in Table 5, where the results are presented in mean±S.E.M. (standard error of the mean) in Table 5.







TABLE 5





the results of cell viability test of MTT assay

















4G













Blank*
10 μM
20 μM
50 μM
100 μM





Vitality (%)
100
93.45 ± 4.91
87.30 ± 1.93
93.06 ± 4.41
87.81 ± 7.83













Vehicle
4GX













controls**
10 μM
20 μM
50 μM
100 μM





Vitality (%)
100.90 ± 4.05
97.18 ± 6.52
100.78 ± 4.06
105.48 ± 2.18
94.31 ± 9.76





*Blank, setting blank as 100.


**Vehicle controls, are presented to demonstrate the cell vitalities are not affected by DMSO






Generally, it is believed that the test substance is substantially non-cytotoxicity when the resulting cell vitalities are more than 80% or 85% after exposuring the cells in an effective concentration of test substance during the survival test. According to the results presented in Table 5, the cell vitalities are all above 85% for both 4GX and 4G with concentration to 100 μM, and thus it is believed that 4GX and 4G are non-cytotoxicity.
Gelatin Zymography
To evaluation the inhibitory effectiveness of gelatinase of 4GX and 4G, a gelatin zymography is needed to be conducted.
The cell suspension solution (with concentration about 5×105 cells/mL) of the previous cell pre-treatment process was placed to a 24-well cell culture plates for 24 hours of incubation at 37° C. Subsequently, 1 μL of the solutions, containing 4GX and 4G, in different concentrations from 10 to 100 μM (10, 20, 50, 100 μM) were applied into each well respectively as vehicle controls, 1 μL of catechin in concentration of 100 μM was applied into the well as a positive control, whereas the well applied without any compounds is as blank, followed by 24 hours of incubation at 37° C. Afterwards, the plate was placed into ice to terminate the reaction, the supernatant of each well were taken out separately for the further experiments.
Supernatants and two volume of a sample loading dye (500 mM Tris-HCL, 25% Glycerol, 10% SDS, 0.32% Bromophenol blue, pH 6.8) and one volume of a dye were mixed together to use as the indicator of running end point. Afterwards, the enzyme activity was analyzed by polyacrylamide gel electrophoresis (PAGE).
1% of gelatin and 10% of polyacrylamide were contained in the gel of PAGE, where gelatin acts as the substrate for gelatinase (pro MMP-2, MMP-2 and pro MMP-9). The running buffer was 25 mM Tris-base, 192 mM Glycerol and 0.1% sodium dodecyl sulfate (SDS), pH 8.3, and the power supply was 125 V/80 mA.
Following the electrophoresis, the gel was washed with 2.5% Triton X-100 at 24° C. twice in 30 minutes to remove the dye and SDS from the gel. Afterwards, the gel was incubated with a reacting buffer (50 mM Tris-base, 200 mM NaCl, 5 mM CaCl2 and 0.02% Brij 35, pH 7.5) at 37° C. for 24 hours in a thermostatic cabinet, enabling gelatinase enzymatically react with gelatin. The fixing solution (7% acetic acid and 40% methanol) was subsequently applied to the gel for 30 minutes, in order to secure the proteins onto the gel. After 30 minutes, the gel was stained with Brilliant Blue G-Colloidial, and then destained with the destain solution (10% acetic acid and 40% methanol) for visualization of the gelatinase reactive positions against the background of the gel.
Finally, the gel was then analyzed by image analysis system (Vilber Lounmat, France), and the analysis software was Bio-1 Dversion 99. Setting the blank as reference, setting as 1, and the other groups are presented in relative values in contrast to blank.
The formula for calculating the inhibitory effectiveness is presented below. The results of gelatin zymography are provided on Table 6, and all values are presented as Mean±S.E.M.

(the values of blank−vehicle controls)×100%








TABLE 6







the inhibitory effectiveness of 4GX and 4G to gelatinase









Inhibitory
4GX
4G













effective-
Pro

pro
Pro

pro


ness (%)
MMP-2
MMP-2
MMP-9
MMP-2
MMP-2
MMP-9





10 μM
15.83 ±
25.46 ±
 3.80 ±
10.13 ±
29.93 ±
18.73 ±



4.48
11.50 
4.8 
5.85
5.99
8.86


20 μM
31.53 ±
63.57 ±
15.83 ±
16.80 ±
48.07 ±
28.70 ±



9.79
4.35
4.48
1.71
3.63
12.54 


50 μM
62.30 ±
76.20 ±
32.17 ±
30.13 ±
73.27 ±
44.13 ±



2.43
6.88
2.77
2.20
7.31
11.32 


100 μM 
82.01 ±
85.07 ±
45.47 ±
39.40 ±
80.47 ±
53.50 ±



3.08
3.78
3.57
6.78
2.19
13.25 


Positive
56.30 ±
86.77 ±
64.30 ±
72.97 ±
85.00 ±
76.07 ±


controls (P)
9.89
11.34 
32.57 
9.76
5.50
3.76


(100 μM)


IC50 (μM)
30.00 ±
16.27 ±
>100
99.63 ±
23.01 ±
>100



6.06
1.63

0.18
4.86





* IC50, half maximal inhibitory concentration)


* Positive controls (P), 100 μM of catechin was applied.






Generally, it is believed that the substance has substantially inhibitory effectiveness when the resulting values of inhibitory effectiveness (%) of the substance are larger than the positive controls. According to the results from Table 6, it indicates that the inhibition activities of 4GX or 4G to MMP-2 are both desirable. The results are comparable with positive controls (100 μM catechin) when 4GX and 4G in the concentration of 100 μM were applied. Besides, the inhibitory activity of the 100 μM 4GX to ProMMP-2 even superior to the results of positive controls (100 μM catechin).
IV. Assay of Collagenase
Collagenase type IV and DQ-gelatin was applied in this experiment, where DQ198 (EnzChek Gelatinase/Collagenase Assay Kit, E-12055) is a kind of fluorescent material, which can bonded with gelatin to form DQ-gelatin. Since the capability of gelatin hydrolysis of collagenase type IV has been demonstrated, DQTM-gelatin can be a substrate for collagenase. Thus, the principle of this experiment is that the activity of collagenase is examined by measuring emission intensity of DQ fluorescent as that the chemical bonding between DQ-gelatin can be enzymatically digested by collagenase.
The fluorescence intensity can be measured in a florescence micro-plate reader equipped with standard fluoresce in filters. Digested product from the DQ-gelatin and DQ collagen substrate has absorption wavelength at 495 nm and fluorescence emission wavelength at 515 nm. Therefore, the activity of collagenase can be determined when the fluorescence emission intensity at 515 nm larger than the blank. In other word, the resulting fluorescence emission intensity would decrease at 515 nm if the collagenase inhibitors were applied. The results of collagenase assay are provided on Table 7, and all values are present as Mean±S.E.M.







TABLE 7







the result of collagenase assay













Positive controls



4G
4GX
(P)













Samples
20 μM
40 μM
20 μM
40 μM
20 μM
40 μM





Inhibitory
52.12 ±
61.73 ±
23.51 ±
47.50 ±
12.80 ±
38.41 ±


effectiveness %
4.30
1.98
2.81
2.90
5.40
1.43





*p, positive controls, 1, 10-phenanthroline were applied.






The inhibitory effectiveness of 4G and 4GX are obvious better than positive controls, and 50% of the inhibitory effectiveness are achieved when 20 μM of 4G were applied according to Table 7. Since 4G and 4GX are able to decrease the activities of collagenase, the prevention of ageing can be achieved by 4G and 4GX.
V. Transdermal Penetration Test
To evaluation the effectiveness of topical use of 4G and 4GX, the transdermal penetration test of 4G was performed.
Franz-type diffusion cell is the apparatus for this experiment, where its structure as a set of vertical double-diffusion and detachable glass containers. The upper donor chamber is a hollow cylindrical for the sample injection, and the bottom of the upper donor chamber has a contacting surface. The lower receptor chamber is double-layered hollow cylindrical diffusion container, where the inner layer of the receptor chamber made of glass contains a fluid for sampling, the outer layer of the receptor chamber is filled with circulating water kept at 37±1° C. for the human body simulation. The contact area between the donor chamber and the receptor chamber is 0.785 cm2 (the actual penetration area), and the intermediate is fixed with nude and white mice (all three) skins (epidermis facing upwards) by metal clips as the in vitro transdermal penetration barriers.
The experimental method is presented below. First, 4G was dissolved in 30 wt % propylene glycol aqueous solution, followed transferring 0.5 mL of the aqueous solution (containing 0.25 mg) of 4G into the donor chamber of the Franz-type diffusion cell, and top of the open-ended donor chamber was covered with Parafilm® tightly, and the inner receptor chamber was filled with 5.5 mL of the buffer solution (30 wt % ethanol, phosphate buffer solution, pH 7.4), with continuously stirring at 600 rpm. 0.3 mL of the buffer solution was respectively sampled at time points of 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 hours, and with complete replacement of 0.3 mL of the buffer solution to keep fluid in the diffusion cell the same volume. Finally, the final compound of 4G accumulated in the fluid of the receptor chamber, was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to investigate the amount of the 4G penetrating through skins and reaching to the blood vessel,
After completing the previous experimental step, the compound, 4G, on the surface of nude and white mice skin were washed with deionized water. Afterwards, the skins were trimmed into the circles with diameter equal to the donor chamber, and following record the skin weight. The skins then placed in homogenizer with 1 mL alcohol at speed 300 rpm for 5 minutes, and followed with centrifugation process at 10000 rpm for 5 minutes. The supernatant was filtered by 0.45 μm PVDF and quantified the content of 4G by LC-MS.
The accumulated amounts of 4G of the fluid were nearly zero in the receptor chamber, the amount of 4G in mice skins are presented on Table 8.







TABLE 8







the amount of 4G in the mince skins











The amount of 4G





in the skins
Average
Percentage of 4G in


Samples
(μg)
(μg)
the mouse skins














Nude
1
1.42
1.17 ± 0.21
27.84%


mice
2
1.02



3
1.09


White
4
8.82
6.07 ± 2.43
74.84%


mice
5
4.23



6
5.16









It is observed that 4GX and 4G are capable of inhibiting gelatinase and collagenase according to the embodiment disclosed herein. Besides, 4GX and 4G can penetrate epidermal layer to basal layer and dermal layer of skin to inhibit the activity of gelatinase and collagenase, and thus mitigation of skin ageing can be achieved.
The reader's attention is directed to all papers and documents which are filed concurrently with this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, each feature disclosed is one example only of a generic series of equivalent or similar features.

## Claims

1. A purified compound 4GX having a chemical formula as follow:

2. A method of inhibiting the activity of MMP-2, Pro MMP-2, Pro-MMP-9, collagenase type IV or any combinations thereof containing, the method comprises:
applying a composition containing an effective amount of a compound 4GX having a formula shown below on skin