Duan Xin,Wu Xuemei,Wu Yongchao,Yang Yu,Hu Changjiang

Sichuan Green Herb Technology Development Co.,Ltd.,China

Abstract The quality standard of Chuanxiong toothpaste was established.HPLC was used to determine the content of ferulic acid in Chuanxiong toothpaste and TLC was used to identify the Chuanxiong in the toothpaste.The content of active ingredients extracted from Traditional Chinese medicine by decocting water was high,and the corresponding toothpaste had a good hemostatic effect.The identification by use of thin layer chromatography(TLC) showed with clear spots and strong specificity for both the test product and the control product (Levistilide A) as observed under ultraviolet light (254 nm) with n-hexane-ethyl acetate (3∶1) as the developing agent,which could be used as the basis for the qualitative identification of Chuanxiong toothpaste.The ferulic acid in the range of 0.019,3～0.481,7 μg mL-1 showed a good linear relationship with the peak area and the recovery rate of sample was between 99.5% and 101.2%.This method was simple,stable and reliable,and could be used as the quality control method for ferulic acid in Chuanxiong toothpaste.

Key words Chuanxiong toothpaste; development; ferulic acid; HPLC; TLC; quality standard

Gingival bleeding is one of the common oral diseases,which refers to a small amount of bleeding caused by spontaneous or slight irritation of the gums.[1]Gingival bleeding is divided into two categories of passive bleeding and active bleeding.Passive bleeding is infiltrated by a large number of inflammatory cells when the gingival is mechanically stimulated the connective tissue of the gingival and the small arteries,capillaries and venules are dilated and congested.Active bleeding is bleeding in the absence of any stimulus and mostly manifests as gingival bleeding triggered by systemic factors,which is extensive,profuse and difficulty in stopping bleeding.[2]Gingival bleeding can be induced by chronic inflammation of the gums.Calculus and plaque are the main factors causing gingivitis.Hence,keeping the mouth clean and inhibiting the growth of oral bacteria is an important measure to prevent gingival bleeding and periodontitis.[3]

Ligusticum chuanxiong Hort belongs to the genus of Ligusticum and the dried rhizome is used as the officinal medicine.Chuanxiong was first found in the Shennong Benzao Jing and has the effects of promoting blood circulation,regulating qi,dispelling wind and relieving pain.Modern phytochemical studies have shown that Chuanxiong is composed of volatile oil,alkaloids,polysaccharides,phenylphthalides,and organic phenolic acids and so on.Pharmacological research revealed that Chuanxiong has the protective effects on cardiovascular and cerebrovascular systems,nervous systems,cardiovascular and cerebrovascular systems,liver and kidney functions,and respiratory systems.[4-7]Chuanxiong as a famous-region drug medicinal material in Sichuan,has developed a variety of functional daily chemical products to meet the diversified development needs of Chuanxiong industrial chain under the drive of market and agriculture.

The acknowledgement of the northern and southern dynasties hongjing Tao recorded that gargling with Chuanxiong extract can treat oral diseases.It can be seen that there are records of Chuanxiong treating tooth diseases in ancient times.In this study,functional toothpaste was developed using the crude drug extracts as raw materials of famous-region drug Chuanxiong and Imperata cylindrica (Linn.) Beauv.[8]Characteristic components of levistilide A and ferulic acid were selected as indexes to establish the qualitative identification and quantitative determination methods,respectively.This study aims to improve the quality control standards of Chuanxiong toothpaste and other related products of Chuanxiong.

1 Experiment

1.1 Instruments and reagents

CAMAG TLC Visualizer-TLC digital imaging system was used to finish the qualitative analysis.Shimadzu LC-20 AD high performance liquid chromatography equipped with photodiode array detector (PDA) (Shimadzu) was used to conducted the quantitative analysis.MS205DM analytical balance equipped with one in ten thousand and XP26 equipped with one thousandth of an analytical balance were provide by Mettler Toledo instruments (Switzerland)and employed to conduct weigh of samples.Ultrapure water machine (Shanghai) was used to provide the water purifier.KQ5200DB type ultrasonic cleaning machine with the power of 600W and 40 KHz(Kunshan) was employed to treat samples.

Reagents of methanol,acetonitrile and phosphoric acid were with the grade of chromatographic pure and provided by SIGMS (USA).N-butanol,sodium chloride,ethyl ether,ethyl acetate and n-hexane were with the grade of analytical pure and supplied by Sichuan Kelong Chemical Limited Company.Reference sample of ferulic acid (C10H10O4) was purchased from China National Institute for Food and Drug Control with purity of >98%.Different batches of Chuanxiong toothpaste samples were provided by Sichuan Green Herbal Science and Technology Development Limited Company.

1.2 Preparation of extracts from Chuanxiong and Imperatae

The medicinal materials of Chuanxiong and Imperatae were weighed proportionally and smashed properly.The mixtures were soaked with 10 times of water for 60 minutes.Then,Chuanxiong and Imperatae were decocted 2 times and 60 minutes for each time.After being filtered,the twice liquid was pumped into a centrifuge tank and concentrated to a clear paste with a density of 1.02 at 65～70 ℃.The clear paste was spray dried at an inlet temperature of 186 ℃ and an outlet temperature of 88 ℃.After ultrafine grinding,the dried powder was mixed with a square cone to obtain the extract of traditional Chinese medicine.

1.3 Development of toothpaste

Weigh each material according to the composition of raw materials in Table 1.Phase A was constitute of glycerin,polyethylene glycol-400,carrageenan,xanthan gum,cellulose gum,hydroxypropyl guar gum.Phase B was made of sorbitol,menthone glycerol ketal.Phase C was form of sodium saccharin,sodium benzoate,tetrasodium pyrophosphate,deionized water.Phase D comprised of hydrated silica.And phase E was constituted of sodium lauryl sulfate,essence and traditional Chinese medicine extract.After heating and dissolving,phase B,C,D and phase E are added into phase A successively.The toothpaste is stirred at a slow speed in the toothpaste machine and continuously for 30～40 minutes under the condition of-0.09MPa vacuum.So that the paste reaches the compact state,and the toothpaste is prepared.

Table 1.Formula of Chuanxiong toothpaste

1.4 Clinical trial of hemostasis of toothpaste

This part was completed in West China Stomatological Hospital of Sichuan University.And the experimental results showed that the use of Chuanxiong toothpaste for subjects for 12 weeks could effectively alleviate gingivitis symptoms,and the subjects showed a good to lerance.[8]

1.5 Quality standard of Chuanxiong toothpaste

Thin layer chromatography conditions were according to the guiding principle of Chinese Pharmacopoeia general rule 0502 (2020 edition).That is,10 μL solutions were absorbed by semi-automatic thin layer pointer on the same silica gel GF254 thin layer plate.Then,the n-hexane-ethyl acetate (3∶1) was used as the developer,and examined under ultraviolet light of 254 nm.

The high performance liquid chromatography conditions were according to Chinese Pharmacopoeia(2020 edition).Agilent C18 column (4.6 mm × 250 mm,5 μm) was used to separate the target material and methanol and 1% acetic acid solution were used as the mobile phase with the ratio of 30:70.The detection wavelength was 321 nm and the flow rate was 1 mL/min.The column temperature set as 30 °C and the injection volume was 10 μL.

Levistilide A reference substance was placed in a brown volumetric flask,and the ethyl acetate was added to prepare 0.1 mg per 1 mL,which was the preparation of TLC identification standard.

The standard preparation was that the standard solution containing 20 μg ferulic acid per 1 mL was prepared by adding 70% methanol into brown volumetric flask.

The preparations of thin layer identification test solution sample were as follow.4 g sodium chloride was placed in a plug cone bottle and 10 mL deionized water was added to shake and mix.Then,20 g toothpaste was weighed accurately and placed into the plug cone bottle,stirred and dispersed evenly.After standing for 10 minutes,100 mL ether was added.The mixtures after stirring and dispersing,ultrasonic extraction were performed for 30 minutes.Filter paper was used to filter the residue and the residue was washed twice by ether with 20 mL each time.Combined filtrate was evaporated in a pan,and dried in a 60 °C water bath.The residue was dissolved with 1 mL ethyl acetate.In addition,the control medicinal of Chuanxiong powder was weighted 1 g,adding ether 20 mL and treating with ultrasonic for 60 minutes.After filtrating,the filter liquor was evaporated to dryness in 60 °C water bath.The residue was adding 2 mL ethyl acetate to dissolve,which was the control medicine solution.

The methods of content detection for sample preparation were according to the following.Sodium chloride was weighted 2 g into plug conical bottle accurately.Then,added into deionized water for 5 mL and shacked mixing evenly.10 g toothpaste samples were accurately weighed and placed in the conical flask.After stirring and dispersing evenly,70 % methanol of 30 mL was added,and the mass was weighed.After ultrasonic extraction for 30 minutes,the sample was taken out and cooled to room temperature,and the mass was weighed again.The sample was centrifuged at 5,000 r/min for 15 minutes and cooled in ice bath water for 10 minutes.Finally,samples were filtered with 0.22 μm microporous membrane and the filtrate was the test solution.

2 Results and discussion

2.1 Thin layer identification of toothpaste

The result for thin layer identification of toothpaste is shown in Figure 1.The test and control product showed with the same color spots in the corresponding position.Besides,the test and control product samples presented 3 spots of the same color in the corresponding position,indicating that the toothpaste contained the indicator component of the Levistilide A.

Figure 1.TLC identification of Chuanxiong toothpaste

2.2 Selection of solvent

According to the method of 1.5,70% methanol,ethyl acetate and chloroform were used as solvent respectively to investigate the extraction solvent of ferulic acid in Chuanxiong toothpaste.The results are shown in Table 2.70 % methanol had the highest extraction rate,followed by chloroform,and ethyl acetate was not detected.Therefore,70% methanol was selected as the extraction solvent for the detection of ferulic acid content in toothpaste.

Table 2.Results by use of different extraction solvents for Chuanxiong toothpaste

2.3 Investigation of linear relationship

Ferulic acid solution was precisely absorbed and injected under the chromatographic conditions in Section 1.5 with the volume of 1 μL,2 μL,5 μL,10 μL,15 μL,20 μL,and 25 μL,respectively.The standard curve was plotted with the peak area as the ordinate and the mass of the reference substance (μg)as the abscissa.The regression equation of ferulic acid was Y=5 205 704.233 4X?2 875.419 5 (R2=1.000,0).The results showed that the linearity of ferulic acid was good in the range of 0.019,3～0.481,7 μg/mL.

2.4 Precision investigation

The precision investigation of ferulic acid was absorbed 10 μL solution and analyzed according to the chromatographic conditions of 1.5 items.The sample was continuously injected for 6 times.The chromatogram was recorded and analyzed.The RSD of peak area was 0.09%,indicating that the precision of the instrument was good.

2.5 Stability experiment

Test solution was precisely taken of 10 μl and injected to analyze at 0 h,2 h,4 h,8 h,12 h,and 24 h under the chromatographic conditions of Section 1.5.The chromatographic peak areas were recorded.In the stability test,the RSD value of ferulic acid content was 0.03%,indicating that the stability of the sample solution was good within 24 h.

2.6 Repeatability experiment

Six test samples of the same product were prepared and determined according to the preparation method of 1.5 items.The RSD of ferulic acid content in the sample was 0.03%,indicating that the repeatability of the method was good.

2.7 Sampling recovery experiment

Six toothpastes were precisely weighed with the weight of 5 g and placed in a conical flask with plugs,respectively.Then,2 g sodium chloride and 5ml deionized water was added.After that,30 mL 70% methanol and 2mL ferulic acid substance with the concentration of 0.034 mg were added into mixtures,respectively.The weight was weighed.After ultrasonic extraction for 30 minutes,the sample was taken out and cooled to room temperature and the weight was weighed again.The sample was centrifuged at 5,000 r/min for 15 minutes and cooled in ice bath water for 10 minutes.The sample was filtered with 0.22 μm microporous membrane and the sample solution was prepared.The reference substance average recovery results of RSD were calculated according to the chromatographic conditions of 1.5 items and the results were shown in Table 3.

Table 3.Results of the recovery test of samples

2.8 Content detection of toothpaste

12 different batches of toothpaste samples were prepared and determined according to the preparation method of 1.5 items.The ferulic acid content detection result is shown in Figure 2 and Table 4.Obviously,the content of ferulic acid in Chuanxiong toothpaste exceeded 0.000 19 %.That is to say,the content of active ingredients in 100 g toothpaste overtopped than 0.19 mg,indicating that this method can meet the analysis requirements of quantitative detection and is suitable for quality control of Chuanxiong toothpaste.

Figure 2.High performance liquid chromatograms of ferulic acid and the toothpaste

Tab.4 Determination results of samples

As a semi-solid test substance,toothpaste is difficult to dissolve in organic solvents due to the influence of matrix.Therefore,sodium chloride is added in the preparation process of the test solution to increase the solubility of ferulic acid in the extract.Chuanxiong contains some small polar substances,which are difficult to be eluted from the chromatographic column during the determination condition.However,continuous injection is easy to cause the retention time of ferulic acid on the chromatographic column to gradually shorten,affecting its separation effect.[9-11]Therefore,methanol is often used to wash the chromatographic column to remove weak polar substances to ensure the reproducibility and chromatographic separation performance of samples.

3 Conclusions

Chuanxiong toothpaste is a daily cosmetic with the certain curative effect,which was developed by Sichuan Green Herbal Science and Technology Development Limited Company.Clinical trials experiments have proved that it has a good preventive effect on gingival bleeding and gingivitis.[8]The main component was Chuanxiong.Hence,Levistilide A was selected as the qualitative detection index,which could quickly identify the components of Chuanxiong in toothpaste.The quantitative determination method of ferulic acid in Chuanxiong was established,which could further improve the quality control standards of Chuanxiong toothpaste and other related products.This study could provide certain reference for subsequent research of daily cosmetic contains traditional Chinese medicine.