Innovation and inheritance: A novel strategy for the development of anti-yellowing cosmetic material from TCM formula

Background Yellow complexion is a major skin issue that bothers Chinese women. The major factor leading to sallow complexion is the accumulation of advanced glycosylation end products (AGEs). Traditional Chinese medicine (TCM) treats dull, yellow complexion as being associated with liver-, spleen- or stomach-related functional disorders. Through analysis of prescription data, we explored combinations of new prescriptions with yellow-dispelling effects. Methods We extracted the most commonly used drug pairs and core medicine in anti-yellowing formulas, then carry out hierarchical clustering analysis and extract new formula combinations. The combination effect was determined by falvnoe, DPPH radical scavenging activity and Non-enzymatic glycosylation inhibiting activity. Toxicological analysis included HET-CAM, 3T3 phototoxicity, multiple irritation and patch test. Result Three empirical formulas were identied according to drug frequency analysis, and a new formula was identied according to the results of hierarchical clustering analysis. A series of biochemical experiments proved that the best anti-yellow-complexion formula combination was the new one. The results of the safety investigation showed that the new formula at a concentration of 5% had no eye irritation effects, no irritant or corrosive effects on mammalian skin, and no adverse reactions with human skin. Conclusion This study explored and analysed the internal laws of TCM for treatment of yellow complexion and, on this basis, developed a plant combination extract material with an anti-yellowing effect on skin. TCM for treatment of yellow complexion and, on this basis, developed a plant combination extract material with an anti-yellowing effect on skin. For the rst time, this study found a new plant composition suitable for use as a cosmetic raw material from a TCM formula and preliminarily explored the methodology for the development of this composition. This study provides a scientic theoretical basis and material selection support for the application of TCM formulas in cosmetics and provides ideas for the modern-day development and application of TCM.


Background
Since 2015, China has vigorously supported the development of traditional Chinese medicine (TCM) and has issued nearly 20 national policies to promote national-level strategies for this purpose. According to statistics from Euromonitor, China's domestic cosmetics market reached 221.072 billion yuan in 2017, with a year-over-year growth of 11.9% (YoY + 5.5 PCT), and has become the second-largest cosmetics market in the world. Compared with the mature Japanese market, it is still too early for China's cosmetics market to hit the ceiling, and the industry certainly has prospects for long-term development. In the past decade, TCM, with its core ingredients, has emerged as a domestic brand in China, and TCM products have received a good market response, gradually becoming the pillar products of pharmaceutical companies. Thus, TCM products are not only suitable for drug development but also exhibit excellent performance in beauty and personal care products.
The application of TCM in the cosmetics industry has the following characteristics: (1) Medicinal plant resources are abundant, and there are more than 300 kinds of plant TCM products included in the 2015 edition of the "list of used cosmetics raw materials" and "Chinese pharmacopeia", which are receiving increasing attention. Many famous local brands use TCM products as the core active ingredients. (2) Several ancient Chinese philosophies, such as yin-yang and the ve elements, or speci c types of individual plants are used as marketing points, and few formulations are constructed under the guidance of theoretical thoughts such as prescription medication in TCM. There is market potential for the development of such products. (3) Drug research and development primarily focuses on e cacy, while cosmetic development should focus rst on safety and then consider e cacy. Both products are heterogenous, and the list of used raw materials should be included with the cosmetics developed. (4) As raw materials for cosmetics, TCMs has broad prospects and considerable market value.
Dullness, lack of transparency, and yellowish complexion are the top keywords associated with Chinese women's awareness of skin problems. Dark yellow complexion is caused by three main factors: (1) Sebum ageing: When the cuticle is dry, the sebum level is low, and the scales produce an irregular surface, reducing the specular re ection of light, so the skin takes on a dark matte appearance. Sebum is removed by cleaning and other means before signi cant amounts of squalene degrade to form a yellow polar compound residue, causing a dark yellow appearance [1] . (2) Food and medicines containing yellow dye and pathological jaundice [2] . Cholecystitis causes yellowing [3] , carotenoids are orange, and melanin is brown. Excessive intake of foods such as oranges and sea buckthorn can lead to temporary yellowing of the skin [4] . This can also be caused by a heterogeneous population of keratin, elastin and connective tissue; lipid metabolism; and abnormal metabolic, in ammatory, or organ dysfunction states [5] . (3) Accumulation of advanced glycosylation end products (AGEs): AGEs in the skin emit characteristic uorescence with a wavelength of 330-500 nm, which is commonly observed in diabetes research [6] . Dyer [7] found that non-enzymatic glycation reactions of collagen and elastin molecules occurred, and the number of AGEs increased with age, leading to yellowing, darkening and decreased visual clarity of the skin. The darkening of the skin has been quanti ed and graded [8,9] . Ageing sebum can be removed by cleaning, and dietary and pathological factors can be avoided, so AGEs are the leading cause of dark yellow facial skin.
TCM has a long history of hair care; it originated in the Qin and Han dynasties, formed in the Sui and Tang dynasties, developed in the Song and Jin dynasties, and was enriched in the Ming and Qing dynasties. Several medicines were developed during these past regimes, and nearly 2,000 TCM hair care formulas were recorded. However, the medicines prescribed by different doctors are affected by the time, climate, geographical location, and speci c conditions of patients, and the dosage and compatibility of prescriptions vary. Therefore, the use of new combinations to analyse the data for historical prescriptions and discover the underlying fundamental rules for combination using the methods and technologies of TCM is a scienti c process. In this study, a new plant composition suitable for use as an effective raw material in cosmetics was proposed based on a classic Chinese medicinal composition, and the methodology for the development of this form was preliminarily explored.
Based on TCM principles, dull yellow complexion is associated with liver-, spleen-or stomach-related functional disorders.
Through data-based analysis of formulas, we explored the a new formula combination with a yellow-dispelling effect. The database used was a Chinese traditional medicine database (Chinese Academy of Chinese Medical Sciences). Two kinds of search words were used according to the description of yellow complexion in TCM classics. The rst word described the appearance directly, and the indirect description was based on textual meaning and pathogenesis. There were 7 search terms used for the rst round of the search.

Formula screening
We found that some formulas unsuitable as cosmetics or for the treatment of yellow complexion needed to be removed. These were summarized into the following ve categories: (1) Associated with severe skin diseases; (2) Associated with short-term body function disorder caused by alcohol abuse or parasites; (3) People in special physiological stages, such as childhood and pregnancy; (4) People who were sallow and emaciated because of major diseases; (5) Retrieved formula was not associated with the yellowing of facial skin.
We also made restrictions on the medicinal materials that constituted the formulas. The rules were as follows: (1) Removal of formulas that contained materials that are not included in the Chinese Pharmacopoeia and had no better alternatives.
(2) Removal of formulas that contained fossils and rare animals. If widely recognized substitutes were available, the formula could be retained.

Formula proofreading
After retrieval and screening,the data were audited, integrated and classi ed. The names of the formulas were checked to ensure that there are no different formulas with the same name and that the same formula was not designate different names. With changes in time, place of origin and accent, the names of medicinal materials may have changed. The standard for Chinese medicinal synonymy in this study was rst compiled according to the 2015 edition of the Pharmacopoeia of the People's Republic of China and then according to the customary names of modern Chinese medicines. A comparison of the ancient and modern names of medicinal materials is shown in Table 1. The name, alias, presence, source, preparation, usage and dosage of the formulas were recorded.

Analysis of formulas
We used the TCM Inheritance Auxiliary System (v 2.5) for formula analysis, which was developed by the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences. The 84 formulas were entered into the system and reviewed by 3 people. The high-frequency drugs and drug combinations in the formulas could be determined by using the software. Then, the compatibility rules of the formulas were summarized, and data-mining algorithms were used to obtain new formulas based on the formula data set.

Extract Preparation
All the herbs were crushed sequentially and passed through a 10-mesh sieve. Empirical formulas were mixed according to the ratios recorded in the classics, and the new formula was mixed with equal proportions. The material-liquid ratio was 1:15; the mixture was soaked for 1 hour and then placed at 80 °C for 2 hours. The extracts were ltered through 0.45-µm pore size lter paper and freeze-dried for 24 hours. Finally, we obtained the formula as a lyophilized powder.

Test for avone
Sodium nitrite-aluminium nitrate-sodium hydroxide colorimetry was used to precisely weigh 0.1 g of the rutin reference substance and dissolve it in 75% ethanol in a 100-ml volumetric bottle with a constant volume and a solution mass concentration of 1.0 g/L. Gradient dilution of the rutin reference substance solution was performed serially with 4 mL of deionized water. Then, 0.3 mL of a 5% (mass fraction) aqueous solution of sodium nitrite was added. After 5 min, 0.3 mL of a 10% (mass fraction) aluminium nitrate aqueous solution was added. Then, 2 mL was taken for a quality score, and after 6 min, 1 mol/L sodium hydroxide aqueous solution and 2.4 mL of deionized water was added, and after 10 min, the absorbance at 510 nm was determined, with 75% ethanol as the control group. The ordinate was used as the absorbance value, and the concentration of the rutin reference substance was used as the abscissa to prepare a standard curve. The solution was prepared by using the freeze-dried powder and deionized water. The absorbance was measured according to the above steps, and the total avonoid concentration of the redissolved liquid was calculated according to the standard curve equation.

DPPH radical scavenging activity
The antioxidant activity of DPPH determined based on the scavenging ability of DPPH free radicals. DPPH ethanol solution (2 × 10 − 4 mol/L), 0.01% vitamin C aqueous solution as the positive control, distilled water as the negative control, and anhydrous ethanol as the control group were used. In tube A, the sample solution was mixed with an equal volume of DPPH solution; in tube B, equal volumes of anhydrous ethanol and DPPH solution were mixed; in tube C, the sample solution was mixed with an equal volume of anhydrous ethanol. After the 30-min light-protected reaction, the absorbance of each tube was measured at 517 nm.

Non-enzymatic glycosylation inhibiting activity
High-pressure steam sterilization experiment supplies and solvents were prepared before the experiment. The reaction solution contained 50 mL of PBS, 0.5 g of bovine serum protein, and 2.7 g of glucose; the sample solution was prepared in sterilized water with gradient dilutions to 2%, 1%, 0.5%; the positive control group was made of aminoguanidine hydrochloride in sterilized water, diluted to 2%, 1% and 0.5%. All the solutions were ltered and sterilized with a 0.2 µm lter membrane. The incubation was performed at 37 °C for 7 days without light, and then, the uorescence intensity was detected at an excitation wavelength of 370 nm and an emission wavelength of 440 nm.   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 avour were analysed ( In the anti-yellowing formulas, warming was the main characteristic. Sweet, pungent and bitter were the main avours. Based on this result, we can infer that nature and avour tended to be gentle. A quarter of the drugs were associated with spleen channel tropism. Then, we connected nature, avour and channel tropism to make a Sankey diagram. We intuitively found that the main nature and avour tended to coordinate the intestines and stomach. 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 lacti ora 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. lacti ora Pall.   (Table 5).

Flavone content
The results of the determination of the total avonoid content of the four groups of lyophilized powders showed that the total avonoid 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. 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 signi cantly higher than that of the Sijunzi decoction and the angelica blood-enriching decoction. 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 avonoid 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. 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 avonoid yield was the highest.

Orthogonal test results
In the orthogonal experiment, the avonoid 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). 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.

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 IC 50 of the tested samples was higher than 1000.0 g/mL, the PIF value was signi cantly 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 speci c results are shown in Table 7.     In addition, most formulas were created in ancient times, when yellow complexion was a sign of malnutrition. However, malnutrition has been replaced by AGEs in modern day. The most typical example is patients with diabetes, which may be understood as another form of overnutrition. Regarding the relationship between yellow complexion and nutrition, it is speculated that the new formula and its secondary metabolites may have an effect on the intestinal ora.

Empirical formulas are still worth to explore on anti-yellow
The three EFs were obtained through the analysis of similar formulas. As far as possible, high-frequency medicinal materials and high-frequency medicinal pairs were included. The combinations were the closest matches to the trends of all the formulas.
Sijunzi decoction [24][25][26] is a tonic supplement that nourishes qi and strengthens the spleen. Danggui Buxue decoction [27][28][29] is a tonic supplement that has a signi cant effect on blood nourishment. Xiaojianzhong decoction [30] is a warming agent that is used for tonifying de ciency and warming in the abdomen and is used to treat liver and spleen de ciency syndrome. The three EFs focus on invigorating qi, invigorating blood, and alleviating the de ciency. Biochemical experiments show that the practical formula Xiaojianzhong decoction has the best effect in the treatment of yellowing, second only to the new formula.

New formula was effective at a range of biochemical experiments
The new formula was also tested by volunteers at a low level after the safety test. A 5% concentration was added to the mask matrix. After mask application, the B* value, which represents the yellow-blue phase of the skin, was signi cantly reduced. We will continue in vivo testing in the future.

This method could be used on application of cosmetic formula and development of cosmetic raw materials
Chinese medicine is a highly practical subject. Due to the large number of studies and classics and because it is not easy to save or retrieve the formulas, there is valuable information that has not yet been obtained. Documents can be digitized for easy storage and retrieval. To date, a great deal of work has been done in the eld of Chinese medicine, and a large number of databases have been established. A large amount of hidden knowledge contained in these databases has not been fully extracted and utilized. The development of data-mining technology will undoubtedly be one of the directions for research on TCM in the future.
There are thousands of TCM cosmetic formulas based on Chinese herbal medicine-avoured ingredients produced in China.
With the rapid development of China's national strength and the prosperity of the beauty and personal care products market, guided by the philosophy of TCM (both internal and external) and the multi-target synergies, prevention and treatment methods for TCM cosmetology, especially via expanded application of past classics, the market share has become extremely impressive.

Conclusion
This study explored and analysed the internal laws of TCM for treatment of yellow complexion and, on this basis, developed a plant combination extract material with an anti-yellowing effect on skin.
For the rst time, this study found a new plant composition suitable for use as a cosmetic raw material from a TCM formula and preliminarily explored the methodology for the development of this composition. This study provides a scienti c theoretical basis and material selection support for the application of TCM formulas in cosmetics and provides ideas for the modern-day development and application of TCM. Sankey diagram of the anti-yellowing formulas' nature, avour and channel tropism  Total avonoid content of the lyophilized formula extract powder Non-enzymatic glycosylation inhibition activity Figure 7 HET-CAM test results showed that the new formula had no eye irritation effect.