Advances in Research of Resources and Cultivation Techniques of Chinese Medicinal Material Radix Bupleuri

Ruyue BAI, Xinyuan GAO, Jinyue WEI, Shiying TANG

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

Abstract At present, with the vigorous development of traditional Chinese medicines, wild Bupleurum chinense resources become increasingly depleted, and its cultivars have become the mainstream of the market. However, due to factors such as a large number of original plants, irregular planting techniques, and poor growth environment, the quality of cultivated B. chinense is uneven and it is difficult to achieve the expected efficacy. In order to protect the wild resources of B. chinense and expand the source of medicinal materials, this paper summarized the research advances of the main resource producing areas and cultivation techniques of B. chinense in combination with relevant documents published at home and abroad, and discussed the future development of B. chinense, to provide a reference for research and sustainable development of B. chinense resources.

Key words Bupleurum chinense, Resources, Distribution of main producing areas, Cultivation, Breeding

1 Introduction

Radix Bupleuri is the dry root of perennial herbBupleurumchinenseDC. orBupleurumscorzonerifoliumWilld[1]. It is slightly cold in nature, bitter in taste, and connects with the liver and gallbladder meridians[2]. It has functions of removing fever and dispersing stagnated liver qi for removing liver qi depression[3]. There are about 150 species ofBupleurum, mainly distributed in the Northwest Plateau and Southwest Plateau. At present, there are 25 species, 8 varieties, and 3 forma in China that are used as Chinese medicinal materials[4]. The main species includeB.chinenseDC.,B.yinchowenseShan et Y. Li,B.scorzonerifoliumWilld.,B.smithiiWolff, andB.microcephalum. Diels,B.bicauleHelm.etc[5].

Radix Bupleuri has a long history of medicinal use in China, and it has high medicinal value, broad prospects and complex components. With the development of extraction and separation technology, a variety of components such as saponins, flavonoids, and volatile oil components have been separated from Radix Bupleuri[6-7]. However, due to factors such as a large number of original plants, irregular planting techniques, and poor growth environment, the quality of cultivatedB.chinenseis greatly different and it is difficult to achieve the expected efficacy. In order to solve the actual problems in production, we summarized the resource situations, main producing areas, chemical components and cultivation techniques ofB.chinense, to provide a reference for the research and development ofB.chinenseresources.

2 Distrbution of resources

As stipulated inChinesePharmacopoeia(2020),BupleurumchinensisDC. andBpleurumscorzonerifoliumWild. are the only original plants of Radix Bupleuri[8]. However, in actual use, some other varieties ofBupleurumare regarded as regular varieties in their local standards. For example, Standards forChineseMedicinalMaterialsinGansuProvincetakesB.smithiiWolff andB.yinchowenseShanetY. Li as regular varieties[9];ChineseMedicinalMaterialsinInnerMongoliatakesB.bicauleHelm. andB.sibiricumvar. jeholense as regular varieties[10]. This is very easy to cause confusion in the source of the commodity Radix Bupleuri on the market, and it is difficult to achieve the expected efficacy.

B.chinenseDC. is distributed in most parts of northern China such as Northeast China and North China. WildB.chinenseDC. is usually grown on sunny hillsides, roadsides or grasses below 1 500 m above sea level[1]. The growth ofB.chinensemust have sufficient sunlight, well-drained sandy loam or humus soil, so that the main root will be strong and the root shape is better. In actual use,B.chinenseis often confused withB.yinchowenseShanetY. Li,B.sibiricumVest,B.marginatumWall. andB.smithiivar. parvifolium[11].

At present, Gansu, Shanxi, Shaanxi and Hebei are the provinces with the largest and most influentialB.chinensecultivation and planting area in China. Longxi County of Gansu Province is a main cultivation area ofB.chinenseand Gansu Province is the province with the highest cultivation yield ofB.chinense[12]. The planting area in Ankang, Lueyang, Shangzhou, Luonan in Shaanxi Province and Longhua and Fengning in Hebei Province are not small. The cultivation ofB.chinensehas brought huge economic benefits to the local area.

According to survey ofB.chinenseresources by Li Xiaoweietal.[13], its planting areas in China are mostly distributed in mountainous areas with an average annual temperature of 3-14 ℃, sufficient rainfall, and an altitude higher than 1 000 m. These conditions are most suitable for the growth and development ofB.chinense.

B.scorzonerifoliumWilld. is mainly distributed in Jiangnan (South of Yangtze River), Northeast, and Northwest of China. The distribution is scattered, and it is used in most parts of the country. In actual use, it is often mixed with fakes such asB.bicauleHelm,B.angustissimum(Franch.) Kitag. orB.komarovianum[11].

In recent years, a large number of scholars havestudied and summarized the germplasm resources and main producing areas ofB.chinensemedicinal materials. At the same time, they have also completed the fourth national survey of Chinese medicinal resources. A survey of wild medicinal materials and cultivation centers in Hebei Province found thatB.chinenseproduced in Chengde City of Hebei Province has a dark color, good medicinal effects, strong fibrous properties, and is not easy to break by hand, and it is very popular in the market. Thus, it can provide a basis for the propagation and reproduction of new varieties ofB.chinensein future.

3 Chemical components

Most of the roots ofB.chinenseare composed of saponins, and also contain a small amount of volatile oil, polysaccharides and other components. The ground parts mainly contain flavonoids, lignans, and coumarins[14-15].

3.1 SaponinsSaikosaponin (SS) is a main pharmacologically active component ofB.chinense, it is a chemical substance extracted from Radix Bupleuri, and its content in stems and leaves is low[16]. Among the extracted saponins, saikosaponins a, c and d have higher content[17]. Among them, saikosaponin a and saikosaponin d are the main biologically active substances, and saikosaponin d has the strongest medicinal properties[16]. The content of saponins inB.chinenseDC. is significantly higher than that of other varieties of the same genus[19], and some plants of the same genusBupleurumdo not even contain saponins. Generally, the content of saponins in the same species ofB.chinensevaries greatly, and the content of saponins in the roots far exceeds that in other parts[20]. Sun Rongetal.separately measured the saikosaponin a content inB.chinenseDC. andB.scorzonerifoliumWilld., and found that under the same factors, saikosaponin a content inB.chinenseDC. is greater than that inB.scorzonerifoliumWilld. They concluded that different original plants have different content of saikosaponin a. Through experiments, Yan Meilingetal.[22]summarized and found that more than 150 kinds of saikosaponins have been found so far, showing that saikosaponins are an important part of the efficacy ofB.chinense.

3.2 FlavonoidsYang Liuetal.[23]found that the flavonols inB.chinenseDC. are mainly divided into three types of aglycones: kaempferol, quercetin and isorhamnetin. Comparing the total flavonoid content ofB.chinense, it can be found that the leaves of theB.chinenseplant have the highest total flavonoid content, followed by the stems, while the root content is very small[20]. Xiao Zongyuetal.[24]isolated and identified salcolin B compounds fromB.chinensefor the first time, and isolated 12 flavonoids fromB.chinenseDC.

3.3 Volatile oilsVolatile oil is also a main medicinal active component ofB.chinense, and it has anti-inflammatory[25], antipyretic and analgesic effects[26]. Ma Xuan[27]held that even the ground and underground parts of the same species ofB.chinensehave a relatively large difference in volatile oil content. Liu Yufa[28]found that the volatile oils contained in different parts of the same batch ofB.chinensewere different, and the content of volatile oil in the roots was twice that of the ground parts. Wu Wei[29]found that the antipyretic effect ofB.scorzonerifoliumWilld was significantly better than that ofB.chinenseDC and through experiments, because the content of volatile oil ofB.scorzonerifoliumWilld is higher than that ofB.chinenseDC. These indicate that different chemical components have different pharmacological effects, and the content of chemical components in stems, leaves and roots is also different. It can be inferred that different medicinal parts ofB.chinensehave different effects, and further experimental analysis is needed to provide theoretical reference.

3.4 PolysaccharidesThe polysaccharides inB.chinensehave the effects of enhancing immunity[30]and lowering blood lipids. At present, the polysaccharides that have been extracted include glucose, galactose, ribose, arabinose,etc[16]. According to findings of Liu Ruetal.[31], the polysaccharide content ofB.chinenseDC. in the same area is higher than that ofB.scorzonerifoliumWilld, they believed that this is caused by genetic and environmental influences. These indicate that genes, environment, and varieties jointly affect the content of polysaccharides.

4 Planting techniques

With the development of the pharmaceutical industry, wildB.chinenseresources can no longer meet market demand. At present, artificially cultivatedB.chinensehas become the main species circulating in the market. Due to factors such as cultivation methods, diseases and insect pests, the yield and quality ofB.chinenseare unstable, which greatly reduces the economic benefits ofB.chinense. Nowadays, how to chooseB.chinensewith good germplasm for cultivation and production and how to ensure the production environment and harvest period have become particularly important.

4.1 Seed propagation and sowing ofB.chinenseSeeds ofB.chinenseare cremocarp and have the characteristics of post-ripening and dormancy. The embryos of newly harvestedB.chinenseseeds are in a dormant state, and the germination rate is low, usually 35%-50%. Treatment is needed to promote later ripening. Dou Qianghong[32]believed that coumarin and saikosaponins are endogenous substances that inhibit the germination ofB.chinenseseeds. Wei Jianheetal.[33]found that the endogenous substance does not exist in the seed coat, and it is not clear where it exists.

Generally, there are two methods for sowingB.chinense, direct sowing and seedling transplanting. Direct sowing is usually carried out in spring in mid-to-late April, and in autumn in early and mid-October. For seedling transplanting, sowing is carried out from early March to late April in spring. Different sowing methods and different sowing periods have a certain degree of influence on the germination rate and yield ofB.chinense. In terms of the sowing period, it can be found through experiments that the spring sowing medicinal materials are higher than summer sowing and autumn sowing in the germination time, germination rate, and seedling rate[34], so spring is the most suitable sowing period forB.chinense. At the time of sowing, deep plowing of the land should be carried out, watering in time to increase the survival rate of seedlings; after sowing, the soil should be covered and compacted, and the soil should be thoroughly watered.

Before the emergence ofB.chinense, one there is drought, a small amount of watering and frequent watering are required to make the seedlings emerge neatly; after the seedlings emerge, the soil should be watered thoroughly, and the soil should be loosened after watering to avoid compaction. During the rainy season, pay attention to drainage. The accumulation of water will cause the roots ofB.chinenseto rot and cause widespread death. Therefore, it is necessary to drain the field water in time during the rainy season. After sowing in spring, when the seedlings ofB.chinensegrow about 6 cm high, the seedlings need to be thinned. When the plants grow to 15 cm high, topping should be carried out in time to provide favorable conditions for the root growth. During the growth peak ofB.chinensefrom June to July, in order to avoid weeds and soil compaction, intertillage and weeding should be used to promote the growth of seedlings, make the main roots stronger, and effectively control the production of branches. Usually,B.chinensesoil should receive three to four times of intertillage during the growth period, especially after drought and rainfall, in order to improve the yield and quality ofB.chinense.

4.2 Diseases and pests and preventionIn the cultivation process, the most common disease ofB.chinenseis root rot, followed by rust and spot blight. After harvesting in autumn, weeds can be cleaned up in time, or soil rotation can be implemented to prevent and control; or chemical agents can be used to effectively control pests and diseases.

Root rot is the main disease ofB.chinenseand it can gradually weaken the plant’s ability to absorb water and nutrients until the roots completely rot, leading to the death of the whole plant. Root rot is mainly manifested by the yellowing and withering of the whole leaves and it usually occurs in the hot summer. Li Yongetal.[35-36]found thatB.chinenseroot rot is common in Beijing. Specifically, the disease of one-year-oldB.chinenseis milder and that of two-year-oldB.chinenseis severer. At the same time, they also found that the same variety cultivated in different regions is quite different in the incidence of root rot and disease index. This shows that the cultivation method and growth environment have a great influence on the incidence ofB.chinenseroot rot. Xiang Qiongetal.[37]surveyed theB.chinenseplanting base in Shangluo of Shaanxi Province, and found that the pathogen causing root rot wasF.solani. Therefore, in the rainy season, it is necessary to pay attention to drainage and flood prevention, and planting on loose soil and well-drained land. For early-onset plants, it is recommended to use chemical fungicides (such as chlorothalonil, thiophanate methyl and methamphetamine) for root irrigation or spray treatment. For early-onset plants, it is recommended to use chemical fungicides (such as chlorothalonil, thiophanate methyl and methamphetamine) for root irrigation or spray treatment. Experiments have shown that 75% chlorothalonil wettable powder (WP) has the best control effect, followed by 80% mancozeb wettable powder. These two agents are broad-spectrum fungicides and have good inhibitory effects on the pathogen ofB.chinenseroot rot[38].

5 Advances in research of breeding technology

For a long time, Chinese traditional cultivation ofB.chinensehas mainly used three methods to obtain seeds from self-retained seeds, wild transplantation and medicinal material market purchases. These methods lead to serious mixed varieties ofB.chinense[39]. With the increase of China’s demand forB.chinensemedicinal materials, the demand for pure, excellent, high-yielding and stable varieties that meet the requirements of theChinesePharmacopoeiais also increasing. In this case, a series of new varieties have sprung up[ 40].

Zhongchai No. 1 was the first domestic and foreign new variety officially recognized by Beijing Seed Management Station for promotion ofB.chinenseDC. It had the obvious characteristics of shallow dormancy, high germination rate, good root shape, stable saponin content and high yield. Yang Chengminetal.[41]studied the dynamic changes of the content of saponins, determined the optimal harvest period and established the theory and technology of large-scale production. Later, on the basis of Zhongchai No.1, Zhongchai No.2 and Zhongchai No.3 were bred through systematic breeding methods, which were improved in quality and yield[42]. The appearance of the Zhongchai series indicates that the quality and traits of new ChineseB.chinensevarieties have been optimized for generations, and the development is good, but its adaptability to certain ecological environments is still weak, and the production situation is not as good as the original production area. In view of this problem, Longxi of Gansu Province bred Longchai No.1, Sichuan Province bred Chuanbeichai No.1 and Chuanhongchai No.1 varieties, but these varieties are prone to delayed growth in Hebei Province, and the effects of poor plant growth are not recognized at the time of purchase by the drug dealer. Therefore, the scientific and technical personnel of She County Agricultural Technology Extension Center and She County Qingyang Seed Industry Co., Ltd. have selected a new variety ofB.chinensefrom the local traditional planting varieties suitable for planting in arid areas Jichai No.1 through joint research, which were identified by Hebei Provincial Science and Technology Achievement Transformation Center in 2017. Since 2018, the Jichai No.1 seed breeding base has been further established and improved, and provided with supporting seed processing and production technology[43].

In recent years, considerable achievements have been made in the molecular breeding ofB.chinense. Through research and experiments, Chen Huaiqiongetal.[44]used the magnetic bead enrichment method to screen out a polymorphic and repeatable Simple Sequence Repeats (SSR) primer for cell DNA marking. Sui Chunetal.[45]established and optimizedB.chinenseISSR-PCR (Inter-Simple Sequence Repeat) reaction system, screened out 30 primers and determined the optimal primer concentration, and constructed the genetic map ofB.chinensewas using ISSR and SSR molecular markers. In terms of genetic engineering, five key enzyme genes in the upstream of the saikosaponins synthesis pathway have been cloned and sequenced[46-48], which provides a basis for the future study of the related functional genomes and physiological phenomena ofB.chinense. The above studies have laid a theoretical and technical foundation for the future use of plant genetic engineering to improve the traits ofB.chinenseand breed new varieties of high-qualityB.chinense.

6 Discussion and prospects

At present,B.chinensehas been successfully planted in large areas, and extensive in-depth studies have been carried out on the effective components and planting techniques ofB.chinense. With the popularization of artificial planting, although the supply demand ofB.chinensehas been alleviated, there is no clear standard for its quality. On the one hand, the state should strengthen the protection of wildB.chinenseresources, establish relevant laws to protect wild resources, conduct in-depth development and utilization ofB.chinense, and invest more manpower and material resources to develop its medicinal value. On the other hand, it is necessary to accelerate the cultivation of good varieties ofB.chinensewith stable characteristics and high content of effective components, and to improve and popularize artificial cultivation techniques. It is necessary to continuously optimizeB.chinensecultivation techniques and use various emerging technologies to solve related problems.

The modernization of traditional Chinese medicine requires the cultivation of good varieties suitable for large-scale standardized cultivation. At present, it is difficult to introduceB.chinensevarieties from different regions to each other, and it is necessary to make further collection, screening and optimization in the later stage.B.chinensebreeding work can be taken from the following aspects: expanding the germplasm resource bank, collectingB.chinensevarieties with different morphological characteristics, and making in-depth exploration of its genetic characteristics[40]; using transgenic technology to obtain characteristic genes, such as resistance to diseases and pests. These can not only increase the yield, but also reduce the use of pesticides. The varieties ofB.chinensebred in this way have the advantages of safe, stable and controllable medicinal material quality, so that the production quality ofB.chinensecan be standardized and industrialized.