3.1Formula retrieval and analysis
3.1.1 Formula retrieal resultes
After retrieval and screening, a total of 84 formulas based on maintenance supplements were obtained. Three were from the Tang period and earlier; 40 were from the Song, Jin and Yuan periods; 28 were from the Ming and Qing periods; and 12 were from the Ming and Qing periods.
3.1.2 Medication rules of anti-yellow formulas
The anti-yellowing formulas involved 173 TCMs. The top 20 medicines with the highest frequency and their frequencies are shown in Figure 1.
Figure 1. High-frequency TCMs in anti-yellowing formulas. Approximately half of the formulas contained four herbs: Indian bread (Poria), Ginseng, Atractylodes and Chinese angelica.
Slightly cold and very cold were included in the cold category, and slightly hot and very hot were included in the hot category. The samples were weighted by frequency, and then, the regularity of nature and flavour were analysed (Table 2-3)
Table2. Nature and flavor of medicines regularity of anti-yellow formulas
Nature and flavour
|
Four nature of drugs
|
Frequency(Times)
|
Rate(%)
|
Four nature
|
Warm
|
468
|
57.71
|
Plain
|
158
|
19.48
|
Cold
|
116
|
14.30
|
Hot
|
61
|
7.52
|
Cool
|
8
|
0.99
|
Total
|
811
|
100
|
Five flavours
|
Sweet
|
469
|
35.61
|
Pungent
|
400
|
30.37
|
Bitter
|
338
|
25.66
|
Sour
|
70
|
5.32
|
Astringent
|
22
|
1.67
|
Salty
|
18
|
1.37
|
Total
|
1317
|
100
|
Table 3. Channel tropism regularity of anti-yellow formula
Channel tropism
|
Frequency
|
Rate(%)
|
Spleen
|
543
|
24.23
|
Lung
|
302
|
13.48
|
Stomach
|
300
|
13.39
|
Kidney
|
299
|
13.34
|
Heart
|
289
|
12.90
|
Liver
|
278
|
12.41
|
Large intestine
|
90
|
4.02
|
Gallbladder
|
56
|
2.50
|
Bladder
|
50
|
2.23
|
Tri-jiao
|
27
|
1.20
|
In the anti-yellowing formulas, warming was the main characteristic. Sweet, pungent and bitter were the main flavours. Based on this result, we can infer that nature and flavour tended to be gentle. A quarter of the drugs were associated with spleen channel tropism. Then, we connected nature, flavour and channel tropism to make a Sankey diagram. We intuitively found that the main nature and flavour tended to coordinate the intestines and stomach.
Figure 2. Sankey diagram of the anti-yellowing formulas’ nature, flavour and channel tropism
3.1.3 Formula rules of anti-yellow formulas
We extracted the most commonly used drug pairs in anti-yellowing formulas and sorted them by frequency (Table 4). Panax ginseng C. A. Mey, Poria cocos (Schw. Wolf, Atractylodes macrocephala Koidz., Angelica sinensis (Oliv.) Diels, Paeonia lactiflora Pall. and Astragalus membranaceus (Fisch.) Bge. merged in pairs formed the commonly used TCM pairs of anti-yellowing formulas. The associated drug network shows that the core of the medicine is P. ginseng C. A.Mey, followed by A. macrocephala Koidz., A. sinensis (Oliv.) Diels and P. lactiflora Pall.
Table 4. CommonlyTCM pairs of anti-yellow formulas
NO.
|
TCM Pairs
|
Frequency
|
1
|
Panax ginseng C. A.Mey & Poria cocos (Schw.)Wolf
|
28
|
2
|
Panax ginseng C. A.Mey & Atractylodes macrocephala Koidz.
|
27
|
3
|
Atractylodes macrocephala Koidz. & Poria cocos (Schw.)Wolf
|
23
|
4
|
Panax ginseng C. A.Mey & Atractylodes macrocephala Koidz.& Poria cocos (Schw.)Wolf
|
19
|
5
|
Panax ginseng C. A.Mey & Astragalus membranaceus (Fisch. ) Bge.
|
17
|
6
|
Atractylodes macrocephala Koidz. & Angelica sinensis (Oliv.) Diels
|
17
|
7
|
Angelica sinensis (Oliv.) Diels & Paeonia lactiflora Pall.
|
17
|
8
|
Panax ginseng C. A.Mey & Angelica sinensis (Oliv.) Diels
|
17
|
9
|
Panax ginseng C. A.Mey & Paeonia lactiflora Pall.
|
16
|
10
|
Atractylodes macrocephala Koidz. & Paeonia lactiflora Pall.
|
16
|
Figure 3. Associated TCM network
3.1.4 Empirical formulas and new formula
A correlation degree of 5 and penalty degree of 2 were set in the system to carry out hierarchical clustering analysis and extract new formula combinations.
According to the 2015 edition of the list of names of used cosmetic ingredients, the new formulas NO.2 and NO.3 contained non-cosmetic beneficial ingredients, so 3 empirical formulas (EFs) and the first new formula (NF) were retained (Table 5).
Table 5. Empirical formulas and new formula (additional file)
3.2 Biochemical efficacy tests results
3.2.1 Flavone content
The results of the determination of the total flavonoid content of the four groups of lyophilized powders showed that the total flavonoid content of the new formula extract calculated by the software program was approximately three to six times higher than that of the three groups of experimental formulas.
Figure 4. Total flavonoid content of the lyophilized formula extract powder
3.2.2 DPPH radical scavenging activity
The results of the determination of DPPH free radical scavenging ability (Figure 5) showed that the new formula had the highest DPPH free radical scavenging activity, followed by the Xiaojianzhong decoction, the activity of which was significantly higher than that of the Sijunzi decoction and the angelica blood-enriching decoction.
Figure 5. DPPH scavenging activity
3.2.3 Non-enzymatic glycosylation inhibiting activity
In the in vitro inhibition of non-enzymatic glycosylation experiment (Figure 6), the new formula showed the most potent inhibition of glycosylation at the three concentrations tested, and the effect was the same as that of the clinically applied drug aminoguanidine at 1.0% and more effective than that at 2.0%.
In the above experiments, among the four groups of freeze-dried powder extracts, the new formula had the best total flavonoid content, glycosylation inhibition activity, and DPPH free radical scavenging ability. According to these results, the optimal composition of the four groups of formula extracts was determined to be the new formula calculated systematically.
Figure 6. Non-enzymatic glycosylation inhibition activity
3.3 Process optimization
3.3.1 Single factor experiment results
By a single factor test, the new formula composition was extracted when the concentration of the ethanol solution was 60%-90%, ultrasonication time was 20 min-40 min, material-liquid ratio was 1:30, and the flavonoid yield was the highest.
3.3.2 Orthogonal test results
In the orthogonal experiment, the flavonoid content and inhibition of the non-enzymatic glycosylation reaction were used as reference indexes, and 30% and 70% weights were included in the comprehensive score, respectively (Table 6).
Y=(Y1/Y1MAX*0.3+ Y2/Y2MAX*0.7)*100
Table 6. Extraction process optimization for the orthogonal experiment
NO.
|
Factors
|
Y1
Flavonoid
(%)
|
Y2
AGEs inhibition(%)
|
Total
Y
|
Material-liquid ratio
|
Time
(min)
|
Ethanol concentration(%)
|
1
|
1:20
|
20
|
70
|
10.95
|
45.60
|
56.43
|
2
|
1:20
|
30
|
80
|
9.04
|
78.69
|
77.74
|
3
|
1:20
|
40
|
90
|
8.94
|
87.10
|
83.92
|
4
|
1:30
|
20
|
80
|
12.03
|
78.47
|
83.53
|
5
|
1:30
|
30
|
90
|
12.98
|
92.24
|
95.87
|
6
|
1:30
|
40
|
70
|
8.45
|
55.15
|
58.70
|
7
|
1:40
|
20
|
90
|
15.05
|
84.89
|
94.42
|
8
|
1:40
|
30
|
70
|
6.91
|
61.95
|
60.79
|
9
|
1:40
|
40
|
80
|
10.40
|
83.27
|
83.92
|
K1
|
218.09
|
234.38
|
175.92
|
|
|
|
K2
|
238.10
|
234.40
|
245.19
|
|
|
|
K3
|
239.13
|
226.54
|
274.22
|
|
|
|
R
|
21.04
|
7.86
|
98.30
|
|
|
|
Optimal conditions: A3, B2, C3
|
|
|
|
The optimal extraction conditions were 90% ethanol solution, a material-liquid ratio of 1:40, and ultrasonic extraction for 30 min. The solid-liquid ratio of 1:30 or 1:40 and different ultrasonication times had little effect on the flavonoid extraction rate and on the inhibition of the non-enzymatic glycosylation reaction. When the volume fraction of ethanol reached 90%, the end product could not be uniformly added to the cosmetic formula. Based on the experimental results and practical application, the final extraction conditions were determined to be as follows: ethanol solution with a volume fraction of 80%, a material-liquid ratio of 1:30, and ultrasonic extraction for 30 min.
3.4 Safety tests result
3.4.1 HET-CAM test result
The positive control chicken embryo showed severe bleeding, blood vessel dissolution, and clotting, while the negative control chicken embryo showed no significant change, and the test system was established. ES=8, and 5% of the new formula compositions showed no eye irritation effect.
Figure 7. HET-CAM test results showed that the new formula had no eye irritation effect.
3.4.2 In vitro 3T3 NRU phototoxicity result
The OD540 values in the solvent control were all greater than 0.4. The positive control was established with IC50 (+UV)=0.6022 μg/mL, IC50 (–UV)=29.91 μg/mL, PIF=49.85, and MPE=0.551, which were in line with OECD standards. The IC50 of the tested samples was higher than 1000.0 g/mL, the PIF value was significantly higher than 2.0 and less than 5.0, and the MPE value was higher than 0.15. According to the test evaluation criteria, the potential phototoxicity of the tested samples was predicted, and the specific results are shown in Table 7.
Table 7. In vitro 3T3 NRU phototoxicity test result. It does not show cytotoxicity when it reaches the highest allowable concentration value (1000.0 μg / mL); + : Expected phototoxicity; – : Expected no phototoxicity.
Control/ Test
|
IC50(μg/mL)
|
PIF
|
MPF
|
phototoxicity
|
No light
|
Light
|
CPZ
|
19.71
|
0.85
|
23.16
|
0.53
|
+
|
Sample
|
>1000.0
|
238.51
|
4.19
|
0.25
|
+
|
3.4.3 Multiple skin irritation test result
Table 8. The results of multipled skin irritation test.
Day
|
Number of animals
|
Irritation response score
|
NF
|
|
Blank
|
Erythema
|
Oedema
|
Totle
|
|
Erythema
|
Oedema
|
Totle
|
1
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
2
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
3
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
4
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
5
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
6
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
7
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
8
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
9
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
10
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
11
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
12
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
13
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
14
|
4
|
0/4
|
0
|
0
|
|
0
|
0
|
0
|
Average scores of per animal and per day
|
0
|
Irritation level
|
No irritation was observed
|
3.4.4 24-hour human body closed patch test
Table 9. 24-hour human body closed patch test result
sample name
|
Observation time(h)
|
Sample concentration(%)
|
Fall off
|
negative(0)
|
suspicious(1)
|
Weak positive(2)
|
Strong positive(3)
|
Extremely strong positive(4)
|
Gel matrix
|
0.5
|
—
|
0
|
27
|
1
|
0
|
0
|
0
|
24
|
0
|
28
|
0
|
0
|
0
|
0
|
48
|
0
|
28
|
0
|
0
|
0
|
0
|
Adding gel
|
0.5
|
5
|
0
|
27
|
1
|
0
|
0
|
0
|
24
|
0
|
28
|
0
|
0
|
0
|
0
|
48
|
0
|
28
|
0
|
0
|
0
|
0
|
According to the results in Table 7-9 and the interpretation of the results in the cosmetic hygiene code (2007 edition), the gel matrix and the gel with 5% composition have no adverse reactions with human skin.
No eye irritation effect of the new composition was detected by testing the chicken chorioallantoic membrane. The phototoxicity test of 3T3 determined that the new structure may have phototoxicity and should not be used in sunscreen products. Through several skin irritation tests, it was determined that the original composition was not irritating or corrosive to mammalian skin. It was determined that the new form had no adverse reaction with human skin by the human closed patch test. Based on the above safety test results, the new formula composition was determined to be safe for the human body and can be used as a cosmetic raw material.