Research Progress on Authenticity Formation of Scutellaria baicalensis Georgi

Ping DONG, Zhongsi LI

Institute of Traditional Chinese Medicine, Chengde Medical University/Hebei Key Laboratory of Research and Development of Traditional Chinese Medicine, Chengde 067050, China

Abstract By analyzing the key factors affecting the authenticity of Scutellaria baicalensis Georgi, this paper focuses on summarizing the research progress of environmental factors (light, temperature, water content, soil), genetic factors and other factors, in order to ensure the quality of S. baicalensis Georgi, find potential suitable areas of S. baicalensis Georgi, and further promote the sustainable development of Chinese medicine resources.

Key words Scutellaria baicalensis Georgi, Authenticity, Environment

1 Introduction

Authenticity is a specific term to identify the quality of Chinese herbal medicines. Authentic medicinal materials refer to high-quality authentic medicinal materials with specific geographical producing areas, the advantages of "high quality, excellent shape and strong effect", standardized production and processing, and recognized by the world[1]. Clarifying the mechanism of authenticity formation of Chinese herbal medicines is the premise of sustainable development of Chinese herbal medicine resources.ScutellariabaicalensisGeorgi is a medicinal plant in the Labiatae family[2]. In China, wildS.baicalensisGeorgi resources are concentrated in North China, Northwest China and Northeast China, and they are most distributed in Hebei, Shanxi, Inner Mongolia, Liaoning, Heilongjiang and other provinces[3]. The authenticity ofS.baicalensisGeorgi is obvious, especially theS.baicalensisGeorgi produced in Chengde, Hebei Province and its surrounding areas has the best quality. In modern times, Chen Renshan compiledDrugIdentification, which recorded thatS.baicalensisGeorgi was produced in Shanxi, Zhili and Rehe (Yanshan Mountain and Hill, Chengde City, Hebei Province), which was the first to explain the existence ofS.baicalensisGeorgi in Rehe[4]. Throughout the distribution changes ofS.baicalensisGeorgi, the overall trend of moving northward may be related to the change of geographical environment.S.baicalensisGeorgi is one of the most commonly used medicinal materials in ready-for-use traditional Chinese medicine. With the increasing demand for Chinese herbal medicines in the market and the depletion of wildS.baicalensisGeorgi resources, the existing resources can no longer meet the demand. Artificially planted and cultivatedS.baicalensisGeorgi has become the main supply source. However, the quality of artificially plantedS.baicalensisGeorgi is uneven, which leads to poor medicinal efficacy[5]. By explaining the scientific connotation of "authenticity", this paper expounds the authenticity formation ofS.baicalensisGeorgi, and provides effective reference for ensuring the quality and therapeutic effect of Chinese herbal medicines and further development and utilization ofS.baicalensisGeorgi.

2 Characteristics of S. baicalensis Georgi

Chengde City (115°54′-119°15′ E, 40°11′-42°40′ N) is located in the northeast of Hebei Province, and in the area where the Greater Khingan Mountains connect the northern Yanshan Mountains to the southwest. It is an important node connecting Beijing, Tianjin, Hebei, Liaoning and Inner Mongolia, the former capital of Rehe Province, and a high-quality producing area ofS.baicalensisGeorgi[6-7]. TheS.baicalensisGeorgi produced in Rehe area is thick, long and solid, golden yellow after removing the outer skin, and the quality is the best. Chengde area is considered as the authentic producing area ofS.baicalensisGeorgi, so theS.baicalensisGeorgi produced in Chengde is known as "Rehe Huangqin".S.baicalensisGeorgi has two specifications, Ziqin and Kuqin, which can treat different symptoms. Kuqin "purges the fire of stomach and lung", while Ziqin "purges the fire of large and small intestines"[8]. Zhao Jiawen[9]proved that Ziqin was better than Kuqin in treating damp-heat syndrome of large intestine. The main effective components ofS.baicalensisGeorgi are baicalin, wogonin, baicalein, wogonin, oroxylin A, chrysin, scutellarin and other chemical components. TheChinesePharmacopoeiastipulates that baicalin is the content detection standard ofS.baicalensisGeorgi, and the content of baicalin shall not be less than 9.0%[10]. Data show that the highest content of baicalin and wogonin is in Longhua County, Chengde City, Hebei Province; the highest content of oroxylin A is in Luanping County, Chengde City, Hebei Province; the highest content of chrysin is in Luanping County, Chengde City, Hebei Province[11]. To sum up, it shows that the quality ofS.baicalensisGeorgi produced in Chengde, Hebei Province is the best, which strongly proves that Chengde, Hebei Province is a producing area of authenticS.baicalensisGeorgi.

Besides flavonoids,S.baicalensisGeorgi also contains phenylpropionate glycosides, iridoid glycosides, triterpenes, alkaloids and other components[12]. It has anti-tumor, antibacterial, antioxidant and cardiovascular malformation treatment effects[13]. And some studies have found that the active constituents inS.baicalensisGeorgi have certain therapeutic effects on mental diseases[14].S.baicalensisGeorgi has anti-inflammatory, immunoregulatory, antibacterial and antiviral effects, and it plays an important role in preventing and treating Novel Coronavirus, so it is necessary to further explore the target and mechanism ofS.baicalensisGeorgi in preventing and treating Novel Coronavirus infection[15]. Studies have shown thatS.baicalensisGeorgi produced in Chengde, Hebei Province has better antipyretic, anti-inflammatory and antiviral effects than medicinal materials produced in other regions[16]. With the continuous development of modernization of traditional Chinese medicine, DNA molecular genetic markers, three-dimensional quantitative analysis of tissue morphology, chemical fingerprint and content determination of traditional Chinese medicine, bioavailability evaluation and other technologies have been widely used to evaluate the authenticity of medicinal materials and further reveal the essence of authenticity[17].

3 Key factors affecting the authenticity of S. baicalensis Georgi

3.1 Environmental factors"Authenticity" indicates a specific ecological environment, including temperature, light, water, soil, altitude and other ecological factors[18]. The authenticity ofS.baicalensisGeorgi is the result of the continuous adaptation of medicinal materials to the environment, which may be formed after countless times of environmental stress. Suitable natural conditions, as the preliminary conditions for the formation of traditional authentic medicinal materials, are conducive to the formation of good properties of medicinal materials and the aggregation of internal components[19-20]. Environmental factors are the primary factors affecting the authenticity ofS.baicalensisGeorgi. Orange change with their environment is a typical phenomenon of "homogeneity". The same plant bears fruits in different shape in different areas, which reflects the key influence of ecological factors on the growth and development of plants. Yuan Yuanetal.[21]put forward the model hypothesis that phenotypic characteristics, genotypic characteristics and environmental modification of medicinal materials affect the formation of authentic medicinal materials. Tian Tian[22]also found that ecological factors are significantly related to the quality ofS.baicalensisGeorgi. To sum up, ecological factors have certain influence on the content of effective components in medicinal plants, so it is very important to choose a suitable ecological environment for medicinal plants, in order to improve the quality of medicinal plants, promote the sustainable development of Chinese medicine resources and the innovative development of Chinese medicine industry.

3.1.1Light. Light is the main environmental factor affecting seed germination, plant growth and physiological process ofS.baicalensisGeorgi, and it is also the main energy source for plant photosynthesis, through which plants carry out physiological metabolism and material accumulation. Through statistical analysis, Han Meietal.[23]studied the diurnal variation characteristics of photosynthesis ofS.baicalensisGeorgi (diurnal variation of net photosynthetic rate ofS.baicalensisGeorgi leaves, diurnal variation of stomatal conductance ofS.baicalensisGeorgi,etc.) and its relationship with environmental factors under three different habitats (Linjiang, Changchun and Taonan, Jilin Province). It was found that the diurnal variation of Pn (net photosynthetic rate ofS.baicalensisGeorgi leaves) in three different habitats showed slight photosynthetic "midday depression", which was caused by stomatal limitation of plant leaves. Under different habitat conditions, the environmental factors affecting Pn ofS.baicalensisGeorgi are different. For example, PAR (photosynthetic active radiation) has the greatest effect on Pn ofS.baicalensisGeorgi, while TL (temperature of leaf) has the smallest effect in Linjiang area; in Changchun, Ta (atmospheric temperature) is the most important to Pn ofS.baicalensisGeorgi, and in Taonan area, PAR has the greatest influence on Pn ofS.baicalensisGeorgi, but Ta (atmospheric temperature) has the least influence. Wei Haoetal.[24]analyzed the effects of different altitudes and different light conditions (shady slope and sunny slope) on flavonoids inS.baicalensisGeorgi, and found that with the increasing altitude, the content of other six flavonoids increased except wogonin. The sample size is not large enough, there is no significant difference in flavonoids content ofS.baicalensisGeorgi under different light conditions (shady slope and sunny slope), but the average value of sunny slope is higher than that of shady slope.S.baicalensisGeorgi is suitable for planting on sunny slope.

Light can affect the growth and development of plants through three aspects: light intensity, photoperiod and light quality[25]. Light has no significant effect on the germination stage ofS.baicalensisGeorgi seeds, but has significant effect on the growth process ofS.baicalensisGeorgi. High intensity can accelerate the synthesis of photosynthetic pigment ofS.baicalensisGeorgi, and PAL (phenylalanine ammonia lyase), C4H (cinnamate-4-hydroxylase) and CHS (chalcone synthase) will also increase significantly, and then the secondary metabolites ofS.baicalensisGeorgi will increase, and finally it will improve the quality ofS.baicalensisGeorgi[26-27]. This indicates that light can affect the gene expression of PAL and UBGAT and the role of UBGAT in the accumulation of baicalin. It shows that light can greatly promote the biosynthesis and accumulation of flavonoids inS.baicalensisGeorgi, and these theoretical discoveries will further provide strong support for light to the quality formation of authentic medicinal materials.

3.1.2Temperature. The content of effective components in the roots ofS.baicalensisGeorgi is mainly affected by temperature, and the influence of high temperature is greater than that of low temperature[28]. Hou Yunliangetal.[29]studied the changes of baicalin content inS.baicalensisGeorgi callus at different culture temperatures, and it was found that the content of baicalin was higher at 25 ℃ and lower at 5 ℃ and the high temperature of 40 ℃ was not conducive to the accumulation of baicalin inS.baicalensisGeorgi callus, and the growth ofS.baicalensisGeorgi required suitable temperature.

The average annual temperature in Chengde is between 5 ℃ and 9 ℃, which is beneficial to seed germination and baicalin accumulation. Most chemical components inS.baicalensisGeorgi are positively correlated with temperature. High temperature is beneficial to the accumulation of baicalin and ensures the quality ofS.baicalensisGeorgi.

3.1.3Water content. Water content is one of the important ecological factors affecting the authenticity ofS.baicalensisGeorgi, and it is also an important condition for photosynthesis of medicinal plants. Water content directly affects the growth and development of medicinal plants, the accumulation of biomass and the accumulation of metabolites. SWC (soil water content) is an important environmental factor affecting baicalin content[30]. Zhou Guofuetal.[31]used Maxent model to predict the ecologically suitable area ofS.baicalensisGeorgi, analyzed the correlation between ecological factors and chemical components inS.baicalensisGeorgi, and found that the variability of precipitation had a great influence on the chemical components ofS.baicalensisGeorgi.

S.baicalensisGeorgi is drought-resistant but is not waterlogging-resistant. Excessive water content will cause the root of medicinal materials to rot. The response of medicinal plants to drought stress can effectively promote the biosynthesis of active components of traditional Chinese medicine[32]. Drought is a special water condition. Excessive water content is not good, different germplasm ofS.baicalensisGeorgi in different stages of growth and development requires different water content. When the water condition was 150 mm, short-term drought was not conducive to the accumulation of baicalin in Gansu and Jilin germplasm ofS.baicalensisGeorgi, but after 1-5 d drought, the baicalin content in Hebei germplasm ofS.baicalensisGeorgi increased greatly. With the aggravation of drought, the baicalin content of Hebei germplasm ofS.baicalensisGeorgi also decreased[33]. Moderate drought stress was beneficial to the transfer ofS.baicalensisGeorgi biomass to roots and increased the distribution ratio of root biomass[34].

Chengde City is rich in water resources, and the annual water output of rivers reaches 3.76 billion m3, which ensures the water content needed for the growth ofS.baicalensisGeorgi[35]. Baicalin and wogonin inS.baicalensisGeorgi were negatively correlated with annual precipitation[36]. Less rain in early spring is the key factor affecting the accumulation of active components inS.baicalensisGeorgi.

3.1.4Soil. Soil inorganic elements and soil microorganisms are one of the essential conditions for the formation of effective components of authentic medicinal materials. The content of effective components in Chinese herbal medicines is correlated with soil factors, and the quality of Chinese herbal medicines is affected by soil inorganic elements and soil fertility. Clarifying the correlation between soil factors and the content of effective components of Chinese herbal medicines can help improve the soil and ensure the quality of Chinese herbal medicines[37]. Xie Lixia[38]determined mineral elements, pH, particle composition, aggregate structure, organic matter, humus and cation exchange capacity in rhizosphere soil of Ephedra sinica in Gansu, Ningxia and Inner Mongolia. Through screening of soil factors affecting the quality of Ephedra sinica, the results showed that ephedrine was significantly or extremely significantly correlated with soil pH, aggregate structure, humus, mineral elements P, K, Mg, Mn, Zn and Sr, and pseudoephedrine was significantly correlated with humus and cation exchange capacity. This proved that the quality formation of medicinal plants was related to soil factors.

Most of Chengde area has mountainous landform, and a small part of plain. The main types of soil-forming parent materials in Chengde area are river and lake alluvium, aeolian loess and so on. The alluvial land of rivers and lakes has loose and fertile soil, which is very beneficial to plantingS.baicalensisGeorgi. Sandy loam soil with slightly alkaline pH also provides necessary conditions for the formation of authenticS.baicalensisGeorgi[39].

S.baicalensisGeorgi needs nutrient soil as its growth condition, and most of the nutrients in soil are provided by nitrogen, phosphorus and potassium. The total absorption and accumulation of nitrogen, phosphorus and potassium byS.baicalensisGeorgi plants is as follows: nitrogen is the highest, followed by potassium and the lowest is phosphorus. Fertilization makesS.baicalensisGeorgi tall and upright, and helps to improve the flavonoids content and the yield of medicinal materials[40].

Not only inorganic elements in soil are beneficial to the growth ofS.baicalensisGeorgi plants, but also microorganisms contained in soil are beneficial to the accumulation of effective components ofS.baicalensisGeorgi. Soil microorganisms include rhizosphere soil microorganisms and non-rhizosphere soil microorganisms. The quantity of these two microorganisms is quite different, and the quantity of rhizosphere microorganisms is much larger than that of non-rhizosphere soil microorganisms. Rhizosphere microorganisms can be divided into three types: growth-promoting bacteria, pathogenic microorganisms and human opportunistic pathogens, and the number of growth-promoting bacteria is the largest[41]. Not all rhizosphere microorganisms are beneficial to the growth and development of medicinal plants, among which the bacteria beneficial to the growth of medicinal plants are called growth-promoting bacteria, including nitrogen-fixing bacteria, phosphorus bacteria,etc.These bacteria form a symbiotic relationship with medicinal plants and promote the metabolism of medicinal plants[42]. Continuous cropping of wheat andS.baicalensisGeorgi can increase the quantity of soil microorganisms, ensure soil fertility and promote the growth ofS.baicalensisGeorgi plants. It was found that the total quantity of soil fungi and bacteria in wheat -S.baicalensisGeorgi rotation land was the largest[43]. Quantitative characteristics of soil microorganisms are important indicators to indicate soil quality and measure soil fertility[44]. Soil microorganism is an important part of soil ecosystem, and it is an essential biological factor to regulate soil ecological processes such as litter decomposition, carbon and nitrogen mineralization, soil nutrient transformation and circulation[45]. Rotation and continuous cropping have great influence on the quantity of soil bacteria, fungi and actinomycetes, and rotation is beneficial to the diversity and stability of soil microbial community and the improvement of soil ecological environment[46]. In Chengde area,S.baicalensisGeorgi is generally planted in rotation mode, which can not only ensure soil fertility, but also increase the economic benefits of growers.

3.2 Harvest timeThe harvest time is also "regional". The harvest time ofS.baicalensisGeorgi in different regions is different, which is caused by different geographical and climatic conditions. The content of effective components ofS.baicalensisGeorgi will change with seasons. The 2020 edition ofChinesePharmacopoeiastipulates that the harvest time ofS.baicalensisGeorgi is spring and autumn. The proportion of chemical components ofS.baicalensisGeorgi harvested in autumn is obviously in a dynamic process with the increase of years, while that ofS.baicalensisGeorgi harvested in spring is in a relatively static state[47]. By comparing the content of baicalin in the roots ofS.baicalensisGeorgi in different harvesting periods, it was concluded that the content of baicalin in the roots ofS.baicalensisGeorgi harvested in June in Chengde area was the highest, and the content of baicalin generally showed a downward trend from June to October, which proved that the best time to harvestS.baicalensisGeorgi in Chengde area was June[48]. Jin Weirongetal.[49]found through field investigation that the harvest time ofS.baicalensisGeorgi in Shandong is autumn. At the same time, it was proved by experiments that the content of baicalin, wogonin, scutellarin and wogonin inS.baicalensisGeorgi harvested in September was the highest, which proved that the best harvest time ofS.baicalensisGeorgi in Shandong was autumn. The content of flavonoids inS.baicalensisGeorgi in different harvesting seasons in Chengde area of Hebei Province was determined. The results showed that the quality ofS.baicalensisGeorgi harvested in spring was obviously better than that harvested in autumn[50]. Determining the harvest season ofS.baicalensisGeorgi can ensure the high quality ofS.baicalensisGeorgi harvested in a suitable time.

3.3 Genetic factorsGenetic factors are the internal factors for the authenticity formation ofS.baicalensisGeorgi. There is obvious genetic variation in the population ofS.baicalensisGeorgi, and there are great differences in flower color, stem shape, stem color and other features for the variedS.baicalensisGeorgi. The important factor affecting the geographical distribution and genetic variation of plant population is the gene flow of plants, and the gene is determined by planting seeds and spreading pollen. Different plants have different gene flow patterns[51]. Chai Junwenetal.[52]used ISSR molecular marker technology to analyze the genetic variation relationship betweenS.baicalensisGeorgi plants at DNA molecular level, revealing that there is great variation between diploidS.baicalensisGeorgi and autotetraploid. The shorter the genetic distance, the more similar the species are. Popularizing the extensive planting of tetraploidS.baicalensisGeorgi is of great significance for maintaining the sustainable development of traditional Chinese medicine resources.

4 Conclusion

Studying the authenticity ofS.baicalensisGeorgi is a complex and comprehensive process. According to the connotation of authenticity ofS.baicalensisGeorgi, combined with textual research of herbs, the influencing factors were analyzed from internal factors (genetic factors) and external factors (environmental factors), and the two major factors cooperated with each other to form a complete system. The study on the influencing factors of authenticity ofS.baicalensisGeorgi is of great significance for discovering the potential suitable growth area ofS.baicalensisGeorgi, studying the development and utilization of related species ofS.baicalensisGeorgi, providing resource guarantee for the protection ofS.baicalensisGeorgi resources and the development of new drug sources, thus forming authenticity with obvious regional characteristics. In the long-term introduction and domestication process, people have summed up planting experience ofS.baicalensisGeorgi belonging to their own areas, thus forming the authenticity ofS.baicalensisGeorgi with obvious regional characteristics.