Quality Evaluation of Atractylodes chinensis(DC.)Koidz.in Chengde Region Based on Dual Component Analysis

Xinyuan GAO, Hongling ZHAO, Ruyue BAI, Zhe LI, Jinyue WEI, Chunying ZHAO

Hebei Province Key Laboratory of Traditional Chinese Medicine Research and Development, Chengde Medical College, Chengde 067000, China

Abstract [Objectives]To evaluate the quality of Atractylodes chinensis(DC.)Koidz.in Chengde region.[Methods]35 samples of A. chinensis from different growth years were collected, and a high performance liquid chromatography(HPLC)method was established for the dual components of atractylodin and atractylone, and the quality evaluation was conducted on the cultivated A. chinensis from Chengde region in terms of moisture, ash and volatile oil content.[Results]The moisture content of all samples met the regulations, and the content of volatile oil was relatively high.Among them, seven samples with atractylodin content lower than 0.3% were unqualified products(accounting for 20%), and one sample had excessive ash content(accounting for 3%).[Conclusions]The established method for the determination of the dual components of Atractylodis Rhizoma is stable, simple, fast, precise and reusable.Besides, according to the Pearson correlation analysis among the indicators, it is concluded that the atractylodin and atractylone content are significantly related to the growth period and the volatile oil content.In addition, the quality evaluation indicators of A. chinensis in Chengde region show that there are differences in the quality of Atractylodis Rhizoma, but the overall pass rate is relatively high.It is necessary to further study the reasons for the differences.

Key words Atractylodes chinensis(DC.)Koidz., HPLC, Correlation analysis, Quality evaluation

1 Introduction

Atractylodis Rhizoma is the dry rhizome of Compositae plantAtractylodeschinensis(DC.)Koidz.orAtractylodeslancea(Thunb.)DC.[1].Atractylodis Rhizoma has a wide range of pharmacological effects, and the pharmacologically active components are mainly volatile oils[2-3], including atractylodin and atractylone and so on.Modern pharmacological studies have found that Atractylodis Rhizoma has antibacterial, anti-inflammatory, anti-cancer, anti-tumor, and nervous system protective effects[4-9].

WildA.chinensismainly grows in Hebei, Inner Mongolia, Shaanxi, Heilongjiang, Jilin, and Liaoning provinces[1].In recent years, wild resources ofAtractylodeslancea(Thunb.)DC have been declining, it becomes an inevitable trend for turning wild into home grown.A.lanceais affected by conditions such as altitude, temperature, rainfall, and soil pH during cultivation[10], thereby affecting yield and quality.In order to ensure the safety of clinical application, the quality evaluation of Atractylodis Rhizoma is particularly important.Some studies have concluded that the germplasm ofA.chinensisin Chengde of Hebei Province is relatively good through the determination and comparison of the effective components[11-12].In view of the authentic production area ofA.chinensis, we evaluated the quality indicators ofA.chinensisin Chengde, such as volatile oil, ash, and moisture, and established a method for the determination of the two main components of atractylodin and atractylone, so as to provide a theoretical basis for the cultivation and development of germplasm resources ofA.chinensisin future.

2 Instruments and reagents

Thermo Fisher UltiMate 3000 ultra-high-performance liquid chromatography system(Thermo Scientific, USA); SQP electronic balance(Sartorius, Germany); SB-5200DT ultrasonic cleaner(Ningbo Scientz Biotechnology Co., Ltd., China); atractylodin reference substance(batch No.19072205, HPLC≥98%, Chengdu Pufei De Biotech Co., Ltd., China); atractylone reference substance(batch No.18110201, HPLC≥98%, Chengdu Pufei De Biotech Co., Ltd., China); ultra pure water(Hangzhou Wahaha Group Co., Ltd., China); methanol, acetonitrile and phosphoric acid are all chromatographically pure.

The 35 batches ofA.chinensissamples were collected from theA.chinensisplanting base, with an altitude range of 200-1 200 m, an annual average humidity of 57%, an annual precipitation of 43-734 mm, and an annual average temperature of 7.8 ℃.All samples were identified by professor Zhao Chunying from as Institute of Traditional Chinese Medicine of Chengde Medical College as dry rhizome ofAtractylodeschinensis(DC.)Koidz.The detailed information was listed in Table 1.

Table 1 Information of Atractylodes chinensis(DC.)Koidz samples

3 Methods

3.1 Determination of the content of active components inA.chinensis

3.1.1Preparation of solution.(i)Preparation of reference solution.Took an appropriate amount of atractylodin reference substance, precisely weighed, and added methanol to prepare 4.52 mg/mL solution, to obtain the atractylodin reference solution; took an appropriate amount of atractylone reference substance, precisely weighed, and added methanol to prepare 1.45 mg/mL solution, to obtain the atractylone reference solution.The prepared atractylodin and atractylone reference solutions were mixed at a certain ratio, and finally a mixed reference solution containing 0.102 7 mg/mL atractylodin and 0.080 6 mg/mL atractylone was prepared.

(ii)Preparation of test solution.Precisely weighed 1.0 g of coarse powder of the samples, placed in a proper conical flask with stopper, precisely added 50 mL of methanol, sealed, weighed, ultrasonic treated(25 ℃, 250 W, 40 kHz)for 40 min, cooled down, weighed again, made up the lost weight with proper volume of methanol, shook up and filtered, took the filtrate, and obtained the test solution.

3.1.2Chromatographic conditions.Chromatographic column: Discovery C18(4.6 mm×250 mm, 5 μm); detection wavelength: 220 nm; detection column temperature: 25 ℃; injection volume: 10 μL; flow rate: 1.0 mL/min; mobile phase:(A)0.2% phosphoric acid water and(B)acetonitrile for gradient elution.The elution conditions are shown in Table 2.Chromatograms were shown in Fig.1 and Fig.2.

Table 2 Gradient elution procedure of mobile phase

Note: No.1 peak is atractylodin and No.2 peak is atractylone.

Note: No.1 peak is atractylodin and No.2 peak is atractylone.

3.1.3Determination of sample content.Took appropriate amount of samples of different batches ofA.chinensis, prepared the test solution of each sample according to the method in Section3.1.1, injected the sample in accordance with the chromatographic conditions in Section3.1.2, and calculated the content of atractylodin and atractylone in samples using the external standard method.The results are shown in Table 3.

Table 3 Determination results of internal indicators of Atractylodes chinensis(DC.)Koidz.

3.2 Methodological evaluation

3.2.1Precision test.Took the mixed reference solution and injected 6 consecutive times in accordance with the chromatographic conditions in Section3.1.2, made a record of the peak areas of atractylodin and atractylone, and calculated the peak areas to getRSD(n=6): 1.05%, 0.09%, respectively, both were than 2.5%, indicating that the precision of the instrument is good.

3.2.2Repeatability test.Took an appropriate amount ofA.chinensissample(BCZ17), and prepared a total of 6 test solutions in accordance with conditions in Section3.1.2, made a record of the peak areas of atractylodin and atractylone, and calculated the component content, theRSDwas 0.87% and 1.05%, respectively, indicating that this method has high repeatability.

3.2.3Linear relationship test.Took an appropriate amount of reference solution of atractylodin and atractylone, added methanol to prepare the mixed reference solution containing 0.102 7 mg/mL atractylodin and 0.080 6 mg/mL atractylone, injected 0.1, 0.5, 1, 5, 10, 15, 20, 25 μL respectively, and plotted the standard curve with the injection volume as the abscissa(X)and the peak area as the ordinate(Y), as shown in Fig.3 and Fig.4.

Fig.3 Standard curve for atractylodin

Fig.4 Standard curve for atractylone

3.2.4Stability test.Took an appropriate amount ofA.chinensissample(BCZ8), prepared the test solution in accordance with the method in Section3.1.1, placed at room temperature for 0, 2, 4, 8, 12, and 24 h, detected in accordance with the chromatographic conditions in Section3.1.2, theRSDwas 0.07% and 0.14%, respectively, both less than 2.0%, indicating that the samples have high stability.

3.2.5Sample recovery rate test.Precisely weighed 0.1 g of 9 pieces of BCZ17 sample with known content of atractylodin and atractylone, added the same amount of reference substance to every three parts as a group, and added the reference solution at the ratio of 1∶0.5, 1∶1, and 1∶1.5 of the sample content respectively.Prepared the test solution in accordance with the method in Section3.1.1, and detected in accordance with the chromatographic conditions in Section3.1.2.Finally, the average sample recovery rate of atractylodin and atractylone was 105.20% and 92.72%, respectively.The results are shown in Table 4.

Table 4 Results of sample recovery rate of atractylodin and atractylone(n=9)

3.3 Determination of volatile oilsThe volatile oils inA.chinensiswere determined using the method specified in Volume 4 ofChinesePharmacopoeia(2020 edition)[13].The results are shown in Table 3.

3.4 Determination of ashesThe ash content inA.chinensiswas determined using the method specified in Volume 4 ofChinesePharmacopoeia(2020 edition)[13].The results are shown in Table 3.

3.5 Determination of moistureThe moisture inA.chinensiswas determined using the method specified in Volume 4 ofChinesePharmacopoeia(2020 edition)[13].The results are shown in Table 3.

4 Results and analysis

We compared 35 samples ofA.chinensisand found that there are indeed differences in the quality ofA.chinensisof different growth years.The the content of atractylodin specified inChinesePharmacopoeia(2020 edition)[13]is an indicator for judging the quality ofA.chinensis.In this study, the content of atractylodin in 35 samples ofA.chinensiswas relatively high.The transplanted wild BCZ30 with five growth years has the highest atractylodin content, which was 0.841 0%.Among them, seven samples with atractylodin content lower than 0.3% were unqualified products, accounting for 20% of all samples.In all samples, the atractylone content was unevenly distributed.The BCZ26 sample had a higher atractylone content.This sample was a wild transplanted strain with five growth years.In the future screening of high-quality germplasm resources, the site of collecting this sample can be marked and observed.Taking the average of the active component content corresponding to different years, we sketched the scatter plot(Fig.5)It can be seen that the content of atractylodin in the samples ofA.chinensiswith 2-6 growth years showed an overall upward trend.With the increase of the year, the content of atractylodin increased, and the content of atractylodin with 3-4 growth years increased slowly.An experiment indicates that[14]atractylodin content inA.chinensiswas positively correlated with growth years, and as the years increase, theA.chinensiscontent would increase.The atractylone content of all samples increased with the growth years, and the increasing trend was more obvious.

Fig.5 Scatter plot for average content of atractylodin and atractylone

In this study, we analyzed the moisture, ash, and volatile oil content of 35 samples ofA.chinensiscollected in Chengde area.The results in Table 3 show that the moisture of the BCZ29 sample was higher than that inChinesePharmacopoeia, and it was an unqualified product.All other samples were qualified.The measured volatile oil content of all samples met the requirements ofChinesePharmacopoeia, and was similar to the results of studies by Ouyang Limin[15].The ash content of BCZ3 was greater than 7%, which did not meet the requirements ofChinesePharmacopoeia.

5 Pearson correlation analysis among indicators of A. chinensis

After processing the data of each indicator, we imported them into SPSS 22.0 software, and performed Pearson correlation analysis on the indicators ofA.chinensis(Table 4).The results showed that there was a significant correlation between atractylodin, atractylone, total volatile oil, and growth years.The correlation coefficient between atractylodin and volatile oil was 0.909, which is a significant positive correlation, and the correlation coefficient between atractylodin and growth years was 0.965, which is also a significant positive correlation.The correlation coefficient between atractylone and volatile oil was 0.986 7, which is a significant positive correlation, and the correlation coefficient between atractylone and growth years was 0.940, which is also a significant positive correlation.The correlation coefficient between volatile oil and growth years was 0.886, which is a significant positive correlation.The content of volatile oil reflects the quality of medicinal materials.Atractylodin and atractylone are the main active components ofA.chinensisand also belong to volatile oil.Therefore, when evaluating the quality ofA.chinensis, it is necessary to evaluate the content of active components, but also necessary to evaluate the content of volatile oil, which is an important reference.

Table 4 Pearson correlation analysis among indicators of Atractylodes chinensis(DC.)Koidz.

There was no significant correlation between the moisture and ash content ofA.chinensisand other indicators.Besides, the moisture in medicinal materials will be affected by the extension of storage time and storage method.Therefore, in quality evaluation, moisture is not taken as the main indicator.

6 Discussion

Atractylodin is the main component of Atractylodis Rhizoma inChinesePharmacopoeia.In order to evaluate the quality of Atractylodis Rhizoma more comprehensively, we established a dial-component detection method for atractylodin and atractylone.Based on the summary of the literature[16-22], we selected the ultrasonic extraction method(40 min)as the extraction method for this experiment.In terms of the ultraviolet wavelength, we compared the peak resolution under the conditions of 203, 220, 270, and 340 nm, and found that the peak resolution result was the best at 220 nm.In addition, we evaluated six mobile phases: methanol-water, methanol-0.1% phosphoric acid water, methanol-0.2% phosphoric acid water, acetonitrile-water, acetonitrile-0.1% phosphoric acid water, and acetonitrile-0.2% phosphoric acid water.The results show that when acetonitrile-0.2% phosphoric acid water was used as the mobile phase, the baseline of these two components was relatively stable, and the peak resolution was better.At the same time of testing the mobile phase and UV wavelength, we selected the injection volume of 5, 10, 15, and 20 μL respectively; compared 3 different flow rates: 0.5, 1.0, and 1.5 mL/min, and found that the flow rate of 1.0 mL/min and the injection volume of 10 μL were most suitable.Through the methodological evaluation, it is found that the method is stable, and the separation degree of atractylodin and atractylone are both greater than 1.5, indicating that the method can simultaneously detect atractylodin and atractylon.

In recent years, Atractylodis Rhizoma has been widely used and the demand has been increasing.It has become an inevitable trend to expand the cultivation scale ofA.lancea.In the cultivation process ofA.lancea, the quality of medicinal materials will be affected by the cultivation environment and cultivation techniques, and there will be quality differences.In order order to ensure the safety of clinical application, it is necessary to evaluate the quality of Atractylodis Rhizoma.In this study, we analyzed the indicators including total volatile oil, ash, moisture, atractylodin, and atractylone ofA.chinensis.Among them, the atractylodin content of 20% of theA.chinensisdoes not meet the requirements ofChinesePharmacopoeia, and most of them have four growth years.Besides, we made a correlation analysis to process the collected 35 samples of related data ofA.chinensisin Chengde area.We analyzed the relationship between these five indicators and the growth years, and found that the content of volatile oil, atractylodin, and atractylone were significantly correlated with the growth years.

In this study, all samples were rinsed with water and dried in a ventilated environment in the early stage of processing, and the epidermis of the medicinal materials was sanded or peeled regularly until the medicinal materials were completely dried and then crushed.Some experiment has shown that[23]half peeled or unpeeled Atractylodis Rhizoma contain high impurities and exceed the standard ash content.In this experiment, all samples were peeled, but there was still one sample having high ash content.These indicate that the mechanized operation in the cultivation of medicinal materials, the use of chemical fertilizers and pesticides, and the pollution of the planting environment will also cause the ash content of the medicinal materials failing to meet the standard[24].Different processing methods are certainly important for the quality of medicinal materials, but the more important is the cultivation environment and cultivation techniques of the medicinal materials.Only by reinforcing the standardized production of Chinese medicinal materials and protecting the cultivation environment can the yield and quality of Chinese medicinal materials of Atractylodis Rhizoma be guaranteed, and the sustainable development and utilization of Atractylodis Rhizoma resources can be guaranteed.