, , , , , , ，

Tianjin Agricultural University, Tianjin 300384, China

Abstract This study aimed to explore an analysis method for baicalin in Scutellaria baicalensis Georgi and make a reference for the analysis of the medicinal value of S. baicalensis cultivated in Jizhou District. The content of baicalin in S. baicalensis cultivated in Jizhou District was determined using high-performance liquid chromatography (HPLC) to judge whether it met the requirements of Chinese Pharmacopoeia; and according to the requirements of the Chinese Pharmacopoeia of 2015 edition, the contents of heavy metals were determined by ICP-MS. The results showed that the contents of baicalin, Cr, Cu, As, Cd and Pb in S. baicalensis planted in Jizhou District were 14.625 05%, 11.360, 12.830, 0.062 315, 0.025 715 and 0.176 65 μg/g, respectively. The content of baicalin in S. baicalensis planted in Jizhou District was much higher than the standard (≥ 9.0%) of Chinese Pharmacopoeia of 2015 edition, and the contents of heavy metals were also below the limit (≤20/1000 000) required by Chinese Pharmacopoeia of 2015 edition.

Key words Scutellaria baicalensis Georgi, Medicinal ingredients, Baicalin, Heavy metal content

1 Introduction

ScutellariabaicalensisGeorgi (Labiatae:Scutellaria) is a perennial herb[1], and its medicinal part is the root after washing and drying. Radix Scutellariae is conical and has a curved shape, with length of about 8-25 cm and diameter of 1-3 cm. The outer surface is dark yellow or brownish yellow. The texture is hard, easy to beak. Radix Scutellariae is bitter in taste and cold in nature, with effects of clearing heat, eliminating dampness, purging fire, detoxifying, stopping bleeding and curing the fetus[2]. It is better than Rhizoma Coptidis in the performance of medicinal antibacterial and does not produce drug resistance.

In recent years, domestic and foreign scholars have conducted in-depth research on the cultivation, chemical composition, pharmacological effects and quality ofS.baicalensis[3]. Baicalin is the main medicinal component ofS.baicalensis. The content of Baicalin is one of the important bases for judging the quality ofS.baicalensis. High-performance liquid chromatography (HPLC) is used more often in the content detection of baicalin inS.baicalensis. Flavonoids are the main medicinal components ofS.baicalensis. People have adopted new technical methods to explore the anti-oxidation and anti-inflammatory mechanisms ofS.baicalensis[4]. Han Yumei[5]found that baicalin had a significant effect on the enhancement of SOD activity of red blood cells, and it can eliminate oxygen free radicals more quickly, preventing vicious cycle. Liu Yupingetal.[6]found that the DPPH free radical scavenging capacity of the extract ofS.baicalensiswas positively correlated with the content of baicalin in the extract. Heavy metal pollution has a great impact on the quality of Chinese herbal medicines. Once the body ingests high levels of heavy metals, they will cause great harm to the body. According to the requirements of theChinesePharmacopoeia, the heavy metal content of 1 gS.baicalensisextract should be less than or equal to 20 parts per million. The study[7]found that there was a significant relationship between the heavy metals contained inS.baicalensisand specific positions[7].S.baicalensishas a long research history, but there is still no in-depth study on the process of action and the mechanism of action. This study aimed to provide a relatively good place for the large-scale cultivation ofS.baicalensisand helpS.baicalensisto be used more efficiently.

2 Instruments and reagents

2.1InstrumentsandequipmentThe used instruments multi-function pulverizer, water bath, round-bottom flask, beaker, volumetric flask, digestion tube, MS, vortex shaker, ultrasonic instrument (power 140 W, frequency 42 kHz) and high-performance liquid chromatography (HPLC) (Tianjin Haishida Biological Center).

2.2ReagentsThe used reagents included baicalin standard, Radix Scutellariae standard, 70% ethanol solution, methanol, ultrapure water, phosphoric acid solution and concentrated sulfuric acid.

2.3SampleThe experimental sample was annualS.baicalensiscultivated in Jizhou District.

3 Experimental methods

3.1ContentdeterminationofbaicalininS.baicalensis

3.1.1Preparation of baicalin standard solution. The baicalin standard was dried in a blast drying oven at 60℃ for 4 h. Then, an accurate amount (accurate to 0.000 1 g) of the dried baicalin standard was placed into a beaker, and added with an appropriate volume of methanol to prepare the standard solution with a concentration of 60 μg/mL.

3.1.2Preparation of test solution. The plants ofS.baicalensiscollected from Jizhou District were washed with ultrapure water several times, and then air-dried in a well-ventilated place. The same part of theS.baicalensisplants was pulverized. An accurate amount (0.3 g) of the powder ofS.baicalensiswas placed in a round-bottom flask, added with 40 mL of 70% ethanol solution with a pipette, placed in a water bath at 90℃ for 2 h, and stood for cooling after the reflux ended. The solution in the round-bottom flask was filtered into a beaker, and the filtrate was filtrate was transferred to a 100-mL volumetric flask and diluted to 100 mL with 70% ethanol solution. An accurate volume (1 mL) of the above solution was transferred with a pipette to a 10-mL volumetric flask, diluted to 10 mL with 70% ethanol solution, sealed and shaken to obtain the test solution.

3.1.3Chromatographic conditions and determination. The chromatographic conditions for the content determination of baicalin were as follows: filler, octadecylsilane chemically bonded silica; mobile phase, methanol-water-phosphoric acid (47∶53∶0.2); detection wavelength, 280 nm; and number of theoretical plates, > 2 500.

An accurate amount (10 μg) of the baicalin standard solution was injected to liquid chromatography. The same amount of the test solution was subjected to chromatographic analysis under the conditions above, and the content of baicalin was obtained.

Content of baicalin (%)= (Concentration of injected sample × Volume of injected sample)/Mass of sample ×100

3.2ContentdeterminationofheavymetalsinS.baicalensis

An accurate amount (0.45 g) of the powder ofS.baicalensiswas placed in a digestion tube. Two replicates were arranged. The same amount of the baicalin standard was placed in another digestion tube as a standard reference. In each digestion tube, 8 mL of concentrated nitric acid was slowly added. The sample powder on the wall of the digestion tube was flushed into the bottom of the digestion tube. Then, each digestion tube was covered with a glass stopper. All the digestion tubes were stood overnight in a fume hood. The cold-treated digestion tubes were placed in a digestion electric furnace at 125℃ for digestion. After the test solution turned clear, the glass stoppers were removed, and the acid was removed till the liquid level in each digestion tube dropped to about 5 mm. Then, the digestion tubes were taken out, stood, and cooled. The solution in each digestion tube was diluted to 25 mL with deionized water. On a vortex shaker, the solution in each digestion tube was mixed. The solution in each digestion tube was poured into a colorimetric hose, and elemental content analysis was performed on the MS.

4 Results and analysis

4.1ContentofbaicalininS.baicalensiscultivatedinJizhouDistrictAs shown in Fig.1, the main peak band of the baicalin standard began to appear at 15′45″. As shown in Fig.2, the main peak band of the test solution also appeared at this time, indicating that the main peak band of the test solution was baicalin. As shown in Table 1, Table 2 and Table 3, the concentration of baicalin in the samples was 44.712 and 44.267 3 μg/mL, respectively; the peak height of baicalin in the samples was 51 929 and 51 871, respectively; the peak height of the baicalin standard was 60 851 and 60 358, respectively; and the peak area of baicalin in the samples was 1 673 757 and 1 657 110, respectively. The relative standard deviation of the data was small, indicating the data had good parallelism and the experiment was more reliable. According to the following calculation formula:

Relative standard deviation (RSD, %) = Standard deviation (SD)/Arithmetic mean of the calculated results

Fig.1ChromatogramofBaicalininScutellariabaicalensisGeorgiplantedinJizhouDistrict

Fig.2Chromatogramofthebaicalinstandard

Table1Chromatographicanalysisofbaicalin

Sample codeMassgVolumemLDilutionfoldsConcentrationug/mLS. baicalensis-10.304 91001044.712 0S. baicalensis-20.303 51001044.267 3

Table2Injectionanalysisofthebaicalinstandard

SampleMassmgStock solutionmg/mLDiluentug/mLInjectionvolume∥μLBaicalin standard5.120.51251.2010

The relative standard deviations of peak height and peak area of baicalin in the samples against those of baicalin in the baicalin standard were 8.9% and 8.1%, respectively, suggesting that the content of baicalin in the sample was close to that of the baicalin standard.

Table3Contentsofbaicalininthesamples

CodeSample namePeakareaPeakheightConcentrationμg/mL1Baicalin standard1 900 66460 85151.2002Baicalin standard1 932 59860 35851.2003S. baicalensis-11 673 75751 92944.7124S. baicalensis-21 657 11051 87144.267

According to the calculation formula of baicalin content, the contents of baicalin in the samples were calculated as 14.664 5% and 14.585 6%, respectively, with an average of 14.625%. TheChinesePharmacopoeiastipulates that in dried sample, the content of baicalin (C11H18O11) should not be less than 9.0%, so theS.baicalensisplanted in Jizhou District met the standard.

4.2ContentsofheavymetalsinS.baicalensiscultivatedinJizhouDistrictAfter digestion, the contents of Cr, Cu, As, Cd, Hg and Pb in the standard and test samples were determined by ICP-MS (Table 4). TheChinesePharmacopoeiaof 2015 edition stipulates that the contents of heavy metals inS.baicalensisshould not exceed 20%, that is, the contents of heavy metals in samples should be less than 20 000 μg/kg. The contents of heavy metals inS.baicalensisplanted in Jizhou District were lower than the standard.

Table4ContentsofsomeheavymetalsinS.baicalensisplantedinJizhouDistrict

5 Conclusions

In this study, the content of baicalin inS.baicalensisplanted in Jizhou District was determined by HPLC, and the contents of heavy metals were determined by ICP-MS. The results showed that he content of baicalin inS.baicalensisplanted in Jizhou District was 14.625 05%, much higher than the standard of theChinesePharmacopoeiaof 2015 edition, and it is at a medium level compared with other producing areas. The contents of Cr, Cu, As, Cd and Pb inS.baicalensisplanted in Jizhou District were 11.360, 12.830, 0.062 315, 0.025 715 and 0.176 65 μg/g, respectively. TheChinesePharmacopoeiaof 2015 edition stipulates that the content of heavy metal per gram ofS.baicalensisshould not exceed 20 parts per million. The heavy metal contents determined by the experiment were far below the required value, so the heavy metal contents ofS.baicalensiscultivated in Jizhou District did not exceed the standard, more secure.

6 Discussion

6.1EffectsofdifferentproducingareasonthecontentofbaicalininS.baicalensisS.baicalensisgrows in the dry and sunny places such as the top of the mountain and roadside. It prefers heat, tolerant to cold, tolerant to drought, and sensitive to flood, so it should not be planted in the land with poor drainage. The soil is dominated by loam and sandy loam, of which the pH is neutral and slightly alkaline. The producing areas ofS.baicalensisare distributed in most provinces and regions of China, and Shaanxi, Shanxi, Gansu, Shandong, and Hebei are the authentic producing areas[8]. Guan Renweietal.[9]found that different germplasm ofS.baicalensishave a high similarity. However, there are differences in the internal chemical composition, and these differences are mainly influenced by the combined effects of gene regulation and environmental factors. Duan Chongyangetal.[10]found that the accumulation of total flavonoids in different parts ofS.baicalensiswas different at different growth stages, and the total flavonoids content in roots was the highest in September.

Jizhou District is located in the northernmost part of Tianjin, and located in the hinterland of Beijing, Tianjin, Tangshan and Chengde. It is the only semi-mountainous county in Tianjin. Jizhou District has a warm temperate semi-humid continental monsoon climate with abundant sunshine, abundant heat, large temperature difference between day and night, distinctive climate, and distinctive seasons. The climatic conditions in Jizhou District are consistent with the growth habits ofS.baicalensis, and they have a good influence on the growth and development ofS.baicalensis. The results of this study showed that the content of baicalin inS.baicalensisplanted in Jizhou District was 14.625%. Although the content met the requirement of theChinesePharmacopoeia, compared with the study of baicalin content in scutellaria of different habitats studied by Tan Anjun[11], it can be found that the content of baicalin in scutellaria planted in Jixian is only moderate.

6.2EffectsofdifferentproducingareasonthecontentsofheavymetalsinS.baicalensisAfter entering the human body, most heavy metals are extremely difficult to exclude the body, causing accumulation of heavy metals in the body. When the accumulation reaches a specific threshold, irreversible damage will be caused to certain organs of the body. In recent years, with the rapid development of modernization of traditional Chinese medicine and the continuous increase in the export of traditional Chinese medicine, the safety of medicinal materials has received widespread attention. The obstruction of export of Chinese herbal medicines due to heavy metals and pesticide residues’ exceeding the standards has been reported continuously, which has become a key issue for the modernization and internationalization of traditional Chinese medicine[12]. TheGeneralPrincipalsandGuidelinesof theChinesePharmacopoeiaof 2015 edition is the guiding principle for all kinds of test samples and test methods. It stipulates the inspection of heavy metals, harmful elements and organochlorine pesticide residue in Chinese herbal medicines. This is of great significance for improving the quality, safety and efficacy of Chinese herbal medicines. TheChinesePharmacopoeiastipulates that the content of heavy metal per gram of sample should be exceed 20 parts per million. TheGreenIndustryStandardsfortheImportandExportofMedicinalPlantsandPreparationspromulgated by the Ministry of Foreign Trade and Economic Cooperation of China stipulates that the contents of Pb, Cd, Hg, and As should not exceed 5.0, 0.3, 0.2 and 2.0 mg/kg, respectively[13].

The content of heavy metal in different parts ofS.baicalensisis different. In different periods, the proportion of heavy metal is different, and the adsorption of heavy metal in the soil is also different. A study[14]showed that the adsorption strength of heavy metal elements in soil byS.baicalensisranked as Cu > Cd > Hg > As > Pb[14]. The contents of Cr, Cu, As, Cd and Pb inS.baicalensisplanted in Jizhou District were 11 360, 12 830, 62.315, 25.715 and 17.665 μg/kg, respectively, lower than the limit required by theChinesePharmacopoeia. They (except that of Cu) were lower than those inS.baicalensiscultivated in Ningxia[15]. The result showed that the contents of heavy metals did not exceed relevant standards, soS.baicalensiswill not cause harm when ingested into the human body. The parallel test was used in this study, and the results showed a good parallelism, indicating that the test results were more reliable.

6.3EffectsofcontentsofbaicalinandheavymetalsinS.baicalensisonthequalityofS.baicalensisWith the development of modern science and technology, it is recognized that most of the medicinal components of plants belong to secondary metabolites, and they are the result of interaction of plants with the environment during long-term evolution[16]. There are many factors affecting the content of heavy metal sin Chinese herbal medicines, such as origin, species and variety, and cultivation measures. Under heavy metal stress, oxidative stress will be induced in plants, causing plant cells to be destroyed, thereby affecting the content of secondary metabolites as anti-stress defense substances in plants.

Baicalin is a flavonoid extracted from the dried roots ofS.baicalensis, which hydrolyzes into baicale and glucuronic acid, and it is one of the main active ingredients ofS.baicalensis. Related study[17]has shown that ionomycin or concanavalin can activate T lymphocytes in the body of white mice and accelerate their propagation. Baicalin products are not only used in the field of natural pure botanicals, but also in the prevention and treatment of human and animal diseases, especially the prevention and treatment of malignant diseases. The application fields of baicalin are very extensive, and its market prospect is broad. At present, it has become a hot spot in research at home and abroad[18]. Many traditional Chinese medicine preparations containingS.baicalensisuse baicalin as a quality control standard. Baicalin has a high content inS.baicalensis, with good biological activity and poor solubility and membrane permeability, and it is a relatively unstable compound[19]. The factors affecting the quality of Chinese herbal medicines are complex and changeable. Different germplasm and ecological environment, as well as chemical composition have a greater impact on the quality of Radix Scutellariae[20].