Optimal Prescription And Quality Evaluation Of Cistanche Whitening Cream

Abstract:

Objective: To select the best prescription of Cistanche whitening cream and determine its quality standard.

Methods: Orthogonalex periment was designed to screen the best prescription; the physical and chemical properties of Cistanche whitening cream were tested, the cistanche in the whitening cream was qualitatively identified by the TLC method, and the Verbascum glycosides in the whitening cream were determined by HPLC.

Results: Through orthogonal experiments, the optimal prescription of whitening cream was determined to be 0.3g stearyl alcohol,1.0g Tween60,0.25gSpan60, and 0.09g carbomerU30. The TLC spots of Cistanche Whitening Cream are clear and specific. The concentration of verbascoside in the range of 6.25~200μg/mL showed a good linear relationship with the peak area.

The average content of verbascoside in Cistanche was 0.142mg/g,0.143mg/g,0.146mg/g,respectively.

Conclusion: The prepared Cistanche Whitening Cream has a good appearance and is the base for multis reasonable; the method of content determination is simple and feasible, with good reproducibility, high precision, and reliable results. It can be used to determine the content of verbascoside in Cistanche Whitening Cream. 

Keywords: Cistanche; Whitening Cream; Prescription Optimization; Preparation Method; Quality Evaluation

Skin color is related to the presence of various biological pigments that help protect against solar radiation, but the most important pigment in determining skin color is melanin [1]. Melanin is produced in melanocytes, which have a lysosomal organelle called melanosomes that synthesize and store melanin [2]. The synthesis of melanin is initiated by L-tyrosine, and under the catalysis of tyrosinase, melanin is generated through a series of complex processes such as dopachrome, dopaquinone and dopa. Among them, the content ratio of 5,6-dihydroxyindole (5,6-dihydroxyindole, DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6-dihydroxyindole-2-carboxylicacid, DHICA) determines the Changes in skin color from black to brown [3-4].

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At present, most of the whitening cosmetics in the market target tyrosinase, and by controlling the activity of tyrosinase, the production of melanin can be reduced to achieve whitening effect [5]. Commonly used skin whitening additives include kojic acid and its derivatives, fruit acid, vitamin C, etc., but these additives all have some adverse reactions.

Cistanche is a traditional Chinese medicine, which has been mainly used for the treatment of male diseases since ancient times. Its main active ingredient is phenylethanoid [6]. In modern research, Cistanche extract includes improving sexual function, anti-aging, anti-oxidation [7], improving learning and memory ability, neuroprotection, immune regulation, anti-fatigue, anti-deficiency. Blood and liver protection and other functions. 

In addition, traditional Chinese medicine extracts as product additives have the advantages of good skin affinity, safety, and reliability compared with chemical additives in modern whitening products. Experiments have shown that phenethyloside can significantly down-regulate the activity of tyrosinase [8]. Therefore, in this study, Cistanche deserticola was used as the main ingredient to prepare a whitening cream, and its prescription was optimized and its quality was evaluated, in order to provide a reference for the development of whitening ingredients in Cistanche deserticola.

1 material of Cistanche Whitening Cream

1.1 Experimental reagents

Stearyl Alcohol; Glyceryl Monostearate; PEG100 Stearate; Cetearyl Alcohol; Shea Butter; Synthetic Squalane; Propylene Glycol; Tween 60; Span 60; Glycerin; Carbomer U30 ( All purchased from Shanghai Yuanye Biotechnology Co., Ltd.); Cistanche deserticola (originating in Inner Mongolia, identified as authentic by Professor Xu Liang of Liaoning University of Traditional Chinese Medicine); Verbasin Standard (batch number: 111530-201007, China Food and Drug Testing Research)

Hospital); chloroform, n-butanol, methanol, formic acid, acetonitrile, sulfuric acid, anhydrous ethanol, phosphoric acid are all analytical reagents, and water is pure water.

1.2 Experimental equipment

FA1004 Electronic Balance (Shanghai Yueping Scientific Instrument Co., Ltd.); DZKW-S-4 Electric Heating Constant Temperature Water Bath (Shanghai Medical Instrument Co., Ltd. Medical Instrument No. 5 Factory); SG3300H Ultrasonic Cleaner (Shanghai Guante Ultrasonic Instrument Co., Ltd.); Silicone Prefabricated Plate G (batch number: 20110905, Sinopharm Chemical Reagent Co., Ltd.); C18 chromatographic column (Yuexu Technology Co., Ltd.).

2 Methods and conclusions of Cistanche Whitening Cream

2.1 Preparation of whitening cream

2.1.1 Whitening cream formula: Stearyl alcohol 0.3g, glycerol monostearate 0.5g, PEG100 stearate 0.25g, cetearyl alcohol 0.1g, shea butter 0.75g, synthetic squalane 1.25g , Span 600.25g,

Carbomer U300.09g, Cistanche deserticola extract 0.5g, glycerin 2.0g, propylene glycol 1.25g, Tween 601.0g, triethanolamine 0.07g.

2.1.2 Method of whitening cream

Stearyl alcohol, glyceryl monostearate, PEG100 stearate, cetearyl alcohol, shea butter, synthetic squalane, and Span 60 were heated and melted on a water bath at 80°C and mixed uniformly as oil. Phase, carbomer U30 was added with water and placed on a water bath at 80°C to heat to swell and dissolve, add Cistanche deserticola extract, then add glycerin, propylene glycol, Tween 60 and mix well as the water phase, add the oil phase to the water phase, stir continuously and emulsify , and add triethanolamine, stir evenly, and let it cool to obtain Cistanche whitening cream.

2.2 Preferred whitening cream prescription

The optimal prescription of Cistanche deserticola whitening cream was screened by orthogonal test. According to the fineness, gloss, viscosity, moisturizing degree and other aspects of the whitening cream, a comprehensive score was carried out, the average value was taken, and the optimal prescription of the whitening cream was selected according to the results.

The optimal prescription of whitening cream is A2B1C2D3, namely stearyl alcohol 0.3g, Tween 601.0g, Span 00.25g,

Carbomer U300.09g. It can be determined from Table 3 that the factor that has the greatest impact on the whitening cream is carbomer U30, which has a significant difference.

2.3 Physical and chemical properties testing

By observing the properties and fineness of Cistanche whitening cream, its pH was determined, and centrifugation, low temperature and stability tests were carried out. Place the prepared sample at -20°C for 1 d, and observe whether there is any

The phenomenon of stratification and particle precipitation [9]. It was continuously placed at room temperature for 30d to observe whether it was stable, whether there were delamination, color changes and other phenomena. Cistanche whitening cream prepared according to the method of "2.1.2" is brown

Color cream with a good appearance, fine and smooth, non-greasy when applied to the skin. The measured pH was (6.90±0.05), which met the requirements of the Pharmacopoeia of the People's Republic of China, and no oil-water stratification appeared in the samples after centrifugation. No delamination and color change occurred after being placed at -20℃ for 1 day and at room temperature for 30 days. The whitening cream of Cistanche deserticola showed good stability.

2.4 TLC identification

Precisely weigh 1 g of Cistanche deserticola extract, add an appropriate amount of methanol, ultrasonicate in an ultrasonic cleaner for 15 min, filter, evaporate the water to near dry state, and dissolve in methanol to obtain the reference medicinal material solution. Take 0.5 g of blank matrix and Cistanche whitening cream, add methanol, dissolve by ultrasonic, centrifuge, and take the supernatant as the negative solution and the test solution respectively.

Pipette 10 μL of each of the above 3 solutions, point them on the same thin-layer plate, and deploy a developing agent in the ratio of chloroform 6: n-butanol 1: methanol 2: formic acid 2 [10], take it out and dry it, spray it with 10 % ethanol sulfate, heated at 105 °C until the spots are clear. It can be seen from Figure 1 that the TLC spots of Cistanche whitening cream are clear and specific, and the blank matrix has no interference.

2.5 Determination of verbascoside content

2.5.1 Chromatographic conditions [11] The chromatographic column is a C18 column (250mm×4.6mm, 5μm); mobile phase: acetonitrile-0.1% phosphoric acid solution (20:80); flow rate: 1.0mL/min; detection wavelength: 330nm; column temperature:

30°C; injection volume: 20 μL.

2.5.2 Solution Preparation

①The preparation of the test solution. Precisely weigh 0.5 g of the whitening cream prepared from Cistanche deserticola, add an appropriate amount of methanol, dissolve by ultrasonic for 15 min (40 kHz), and filter it to constant volume in a 10 mL volumetric flask to obtain the test solution. ②Preparation of reference solution. Accurately weigh 4.13 mg of Verbasin reference substance, put it in a 100 mL volumetric flask, dilute to volume with 50% methanol, and shake well to obtain the reference substance solution.

③Negative note: 1. Control medicinal material; 2. Blank matrix; 3, 4, 5 Cistanche whitening cream.

Solution preparation. Precisely weigh 0.5 g of the prepared blank matrix, add an appropriate amount of methanol, dissolve by ultrasonic, and filter to a volume of 10 mL in a volumetric flask to obtain a negative solution. The above solutions were filtered through a 0.45 μm microporous membrane.

2.5.3 System Adaptability Investigation

Take the test substance, reference substance and negative solution respectively and carry out the determination according to the chromatographic conditions under "2.5.1".

The peak time of verbascoside in the test substance and the reference substance was similar, and the peak was not seen in the negative solution, indicating that the specificity was good, and the negative solution did not interfere with it.

2.5.4 Investigation of Linear Relationship

Absorb the reference substance solution of Verbasidin to prepare standard solution of 6.25, 12.5, 25, 50, 100, 200 μg/mL, and measure under the chromatographic conditions under "2.5.1", taking the concentration as the abscissa and the peak area as the vertical Coordinates, the linear regression equation y=28483x+54316 (R2=0.9994) was obtained, which indicated that the linear relationship between verbascoside and peak area was good in the range of 6.25~200μg/mL.

2.5.5 Precision test

Accurately draw the reference solution, and measure it according to the chromatographic conditions under "2.5.1", the RSD of the peak area of verbascoside is 1.58% (n=6), indicating that the precision of the instrument is good.

2.5.6 Repeatability test

Accurately draw the solution of the test sample and measure it according to the chromatographic conditions under "2.5.1", the RSD of the peak area of verbascoside is 1.81% (n=6), indicating that the method has good repeatability.

2.5.7 Stability test

Accurately draw the test solution that was placed at room temperature for 0, 4, 8, 12, 20, and 24h, and measure it. The RSD of the peak area of verbascoside is 1.04%, indicating that the test solution has good stability within 24h.

2.5.8 Sample addition recovery test

Precisely draw 9 parts of the test solution of known content, add the same amount of Verbasin reference substance to dissolve

liquid, and measure according to the chromatographic conditions under "2.5.1". Each 0.5g, according to "2.5.2 ①" method to prepare the test solution, according to "2.5.1" under the chromatographic conditions for sample injection and determination.

3 Discussion of Cistanche Whitening Cream

Although many enzymes are involved in the production of melanin, tyrosinase and tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (Tyrosinase-related protein-2) , TRP-2) plays an important role in melanin synthesis. Specifically, tyrosinase plays a rate-limiting role in catalyzing melanin synthesis and is the most important way for the development of melanin synthesis inhibitors. Melanosis is known to be regulated by more than 125 different genes [12]. These genes regulate some important biological functions of melanocytes, such as cell differentiation and regulation of pigmentation or melanogenesis. Among them, Microphthamia transcription factor (MITF) is the most important member of known melanocyte development transcription factors [13]. MITF regulates its transcription process by binding to the promoter regions of TYR, TRP-1, and TRP-2 gene expression [14]. Cistanche deserticola phenylethanoid can reduce the content of melanin, significantly inhibit the transcription levels of TYR and TRP-1 genes, and down-regulate the protein expressions of TYR, TRP-1 and TRP-2. In addition, Cistanche deserticola phenylglycoside can also inhibit the MITF mRNA transcription level and down-regulate the protein expression of MITF[15].

The demand for whitening cosmetics in China and Asia is increasing year by year, but the active ingredients in most cosmetics usually contain harmful ingredients. my country is rich in traditional Chinese medicine resources and has high safety. Therefore, this study used Cistanche as raw material to prepare whitening cream. The prepared whitening cream has good appearance, delicate and smooth, stable properties, no greasy feeling when smeared on the skin, and strong skin affinity; the physical and chemical properties of it are tested, and the test results show that it has good stability. The orthogonal test design has the advantages of scientific method, simple operation and remarkable effect. Therefore, the orthogonal test was used to screen the optimal prescription of Cistanche whitening cream. 600.25g, Carbomer U300.09g. The factor with the greatest influence on the whitening cream was determined by ANOVA to be carbomer U30. The determination of verbascoside content by high-performance liquid chromatography is simple and feasible, with good reproducibility, high precision, and reliable results.

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