A Review on the Ethnomedicinal Usage, Phytochemistry, and Pharmacological Properties of Gentianeae (Gentianaceae) in Tibetan Medicine

Gentianaceae is a large plant family and is distributed worldwide. As the largest tribe in Gentianaceae, Gentianeae contains 939–968 species, and the Qinghai-Tibet Plateau and adjacent areas are the main centers of diversity for Gentianeae. Species from the Gentianeae are widely used in traditional Tibetan medicine. In this review, a systematic and constructive overview of the information on botany, ethnomedicinal usage, phytochemistry, and pharmacological properties of Gentianeae in Tibetan medicine is provided. The results of this study are based on a literature search, including electronic databases, books, websites, papers, and conference proceedings. Botanical studies showed that Gentianeae includes the subtribe Gentianeae and Swertiinae, and several new genera and taxa have been identified. Approximately 83 species from Gentianeae were used in Tibetan medicine, among which Gentiana and Swertia constituted the largest number of species with 42 and 24 species, respectively. The species from Gentianeae are mainly used as Bangjian (སྤང་རྒྱན།), Jieji (ཀྱི་ལྕེ།), Dida (ཏིག་ཏ།), and Ganggaqiong (གང་གྰཆུང་།) in Tibetan medicine with different clinical applications. More than 240 formulas were found containing Gentianeae species with different attending functions. Phytochemical studies showed that the main active components of Gentianeae species are iridoids, xanthones, flavonoids, and triterpenoids. The bioactivities of plants from Gentianeae include hepatic protection, upper respiratory tract protection, joint and bone protection, glucose regulation, antibacterial, antioxidant, anticancer, and antiviral effects. This review will provide a reference for future research on natural resource protection, plant-based drug development, and further clinical investigation.


Introduction
Tibetan medicine has a long history of being rich in active components, which embodies the precious experience of Tibetans in their long-term struggle against diseases. Moreover, Tibetan medicine is based on a unique theoretical system and strong national characteristics. The origin of Tibetan Medicine is extremely complicated. Tibetan medicine is not only a syncretism of Chinese, India, and Persia medicinal opinions but also has unique historical origins, which may stem from pre-Buddhist sources or may have Ayurvedic origins [1][2][3]. In 2006, Tibetan medicine was included in the first batch of the national intangible cultural heritage list of China.
The history of Tibetan medicine dates back to 200 BC (the reign of Nyatri Tsenpo [གཉའ་ ི ་བཙན་པོ །], the first governor of Tibet). The famous assertion of Tibetan medicine, "Poisonous, there is medicine" was proposed, which foreshadowed the budding of Tibetan medicine [4]. In 641 BC, the governor of Tibet, Songtsen Gampo ( ོ ང་བཙན་ མ་པོ ), married traditional use of this tribe and elucidating the chemical composition and pharmacological effects of Gentianeae will provide a powerful reference for the in-depth study, rational use, and effective protection of this valuable resource.

Data Collections
All data in this review were summarized from references, including scientific journals, book chapters, or dissertations. All the references were searched in several electronic databases, including PubMed, Web of Science, Scopus, ScienceDirect, CNKI (https:// oversea.cnki.net/index/), Google Scholar (https://scholar.google.com/), and Baidu Scholar (https://xueshu.baidu.com/) with "Gentianeae (Gentianaceae) in Tibetan Medicine" as keywords without any other restrictions. Subsequently, literature closely related to botanical study chemical composition, traditional uses, and pharmacological properties were screened. In addition, the classification and geographical distribution of Gentianeae plants were searched in Plant Plus of China (https://www.plantplus.cn/cn), Global Biodiversity Information Facility (GBIF, https://www.gbif.org/) and The Plant List (http://theplantlist. org/). The last accessed date to the links mentioned above in order to acquire data was 31 May 2021.

Botanical Studies of Gentianeae
Gentianeae has the largest number of species amounting to 939-968, which account for about 57% of Gentianaceae [16]. Gentianeae includes the subtribe Gentianeae and Swertiinae. The morphology, palynology, flower anatomy, and chromosome characteristics of Gentianeae were comprehensively summarized, and the classification history was analyzed [16,19,20]. On the basis of classical morphological classification, phylogenetic studies of Gentianeae have made a lot of progress with the development of molecular systematics, and the phylogenetic relationships among groups have become increasingly clear. Several new genera and taxa have been identified.

Subtribe Gentianeae
Struwe et al. divided the subtribe Gentianeae into three genera, Gentiana, Crawfurdia, and Tripterospermum based on the analysis of trnL-intron, matK, and ITS sequences combined with morphological studies [16]. Ho et al. published a new genus, Metagentiana, based on morphological characteristics [21]. Faver et al. separated the genus Sinogentiana from Metagentiana and the genus Kuepferi from Gentiana based on morphological characteristics and molecular phylogeny [22]. Thus, the current subtribe Gentianeae consists of six genera, namely, Gentiana, Metagentiana, Kuepferia, Crawfurdia, Sinogentiana, and Tripterospermum. Gentiana (350 species) is the largest genus of Gentianaceae that originated in the Qinghai-Tibet Plateau and differentiated with the rise of the plateau and with climatic changes [18]. Kuepferia (14 species) and Sinogentiana (2 species) prefer cool and dry habitats and are rather conserved niches. Despite a tendency for niche evolution, Crawfurdia (18 species) and Metagentiana (14 species) are probably restricted to a narrow distribution range owing to their poor dispersal ability [23]. In contrast, Tripterospermum (18 species) has the broadest niche and thrives under the warmest and wettest conditions [24]. A total of 279 species of the subtribe Gentianeae are distributed in China, including 231 species in Gentiana, 9 species in Metagentiana, 9 species in Kuepferia, 16 [25,26]. Therefore, there are 16 genera in the subtribe Swertiinae, of which 13 genera are native to China, including 3 endemic genera. A total of 131 species of subtribe Swertiinae are found in China, including 11 species in Comastoma, 9 species in Gentianella, 5 species in Gentianopsis, 2 species in Halenia, 2 species in Jaeschkea, 1 species in Latouchea, 3 species in Lomatogoniopsis, 17 species in Lomatogonium, 2 species in Megacodon, 1 species in Sinoswertia, 76 species in Swertia, and 2 species in Veratrilla [17]. In the Qinghai-Tibetan plateau, there are about 98 species of subtribe Swertiinae, including 9 species in Comastoma, 7 species in Gentianella, 4 species in Gentianopsis, 2 species in Halenia, 2 species in Jaeschkea, 3 species in Lomatogoniopsis, 15 species in Lomatogonium, 1 species in Megacodon, 1 species in Sinoswertia, 52 species in Swertia, and 2 species in Veratrilla. Although subtribe Swertiinae can be morphologically divided into two large groups (Rotate and Tubular), molecular phylogeny shows that several groups within subtribe Swertiinae are distinct complex groups [20]. Swertia is the main group of subtribe Swertiinae, whereas other related genera, either monophyletic or symphyletic, are derived from Swertia [27]. There are significant inconsistencies between the morphological taxonomy and molecular phylogeny among the genera of subtribe Swertiinae. The possible explanation is that morphological taxonomy cannot reflect the true evolutionary history, and the most significant reason may be the plasticity of the morphological characteristics [27][28][29].

Ethnomedicinal Usage of Gentianeae in Tibetan Medicine
Gentianeae species are important resources in traditional Chinese medicine and Tibetan medicine [5,30,31]. Approximately 133 species (including varieties) in 17 genera of Gentianeae were used in traditional Chinese medicine, among which Gentiana and Swertia constituted the largest number of species with 60 and 35 species, respectively, accounting for 71% of the species [32]. According to our investigation, approximately 83 species were used in Tibetan medicine (Table 1), including 3 species of Comastoma, 42 species of Gentiana, 3 species of Gentianopsis, 3 species of Halenia, 5 species of Lomatogonium, 1 species of Megacodon, 1 species of Sinoswertia, 24 species of Swertia and 1 species of Veratrilla ( Figure 1). The history of the use of Gentianeae species in Tibetan medicine can be traced back to the 8th century in the book "The Remain of Dunhuang Tibetan Medicine" [33], "Sman-dpyad Zla-bavirgyal-po" [6], and "Dpl Ldn Rgyud Bzhi" [7], in which there are descriptions of the efficacy of Tibetan medicines, such as Bangjian ( ང་ ན།), Jieji ( ི ་ ེ །), Dida (ཏི ག་ཏ།), and Ganggaqiong (གང་ ྰ ང་།), belonging to Gentianaceae. These classical works of Tibetan medicine not only record the use and usage of the medicine but cover the morphological characteristics and habitats of the plants used in traditional Tibetan medicine.
The experience of the usage of "Bangjian" was summarized by Tibetan ancestors. It is extremely complicated to determine the origin of its medicinal materials because of the lack of systematic taxonomic knowledge and detailed description in the classic works of Tibetan medicine [14,37,38]. Herbological studies on the material medica resources of "Bangjian" are, therefore, necessary. After a systematic review and analysis of herbalism and phytotaxonomy in both ancient and modern literature, it is widely believed that G. szechenyii and G. algida (including G. algida var. algida and G. algida var. purdomii) are the two original plants of "Bangjian·Gabao" [39]. Nevertheless, research on the material medica resources of Bangjian·Nabao, Bangjian·Wenbao, and Bangjian·Chabao is extremely difficult as existing standards in the material medica resources are inconsistent [40][41][42]. Several plants in Gentiana, Sect. Monopodiae, Ser. Ornatae were used as the resources of "Bangjian·Nabao", "Bangjian·Wenbao", and "Bangjian·Chabao" (Table 1). These plants are very similar in morphology and have different flowers and colors in different regions, seasons, and habitats. It is impossible for Tibetan doctors and farmers in the field to distinguish medicinal materials based on plant taxonomy; thus, the situation of mixed use arises during production [40,41,43,44]. Although the material medica resources of "Bangjian·Nabao", "Bangjian·Wenbao", and "Bangjian·Chabao" are mixed, their effects are similar. These components are mainly used to treat fever and respiratory diseases, and the mixed components may not affect efficacy. It is particularly important to set more reasonable standards and thoroughly study the material medica resources of "Bangjian".
"Shel Gong Shel Phreng" recorded that "Jieji·Gabao grows in hillside meadows. Its stems are red with thick green leaves that are long and glossy. The flowers are white with many green stripes. The stems are erect with apical flowers like Bangjian" [8]. "Tibetan medicine" considers the abovementioned plant with red stems, white flowers, and green stripes as G. straminea and the plant with erect stems and apex flowers is G. tibetica or G. officinalis [5]. "Bee Sngon" states that "Jieji·Nabao growing in the mountains has morphological characteristics like Jieji·Gabao, with blue-purple flowers and thin leaves" [34]. "Shel Gong Shel Phreng" described that "Jieji·Nabao was like Jieji·Gabao, but the stem and leaves tiled on the ground. The leaves are slightly large and the flowers are white without significant luster" [8]. "Tibetan medicine" considered that the material medica resources of "Jieji·Nabao" are mainly G. dahurica and G.crassicaulis [5]. Moreover, other material medica resources of Jijie were used in actual production [46]. In the Qinghai Province, Tibetan doctors use G. hexaphylla as "Jieji·Gabao". In Tibet, M. stylophorus was used as "Jieji·Gabao·Manba" ( ི ་ ྩ ེ །་དཀར་པོ ་དམན་པ) [47]. In addition, G. waltonii, G. robusta, and G. siphonantha were also used as "Jieji" [47].

Dida (ཏི ག་ཏ།)
"Dida" is one of the most representative medicines used in Tibetan medicine. "Shel Gong Shel Phreng" recorded that "Dida" has the effect of purging the liver and gallbladder, promoting diuresis, renewing the physique, and stanching bleeding [8]. Currently, "Dida" is widely used in the treatment of acute jaundice hepatitis, viral hepatitis, cholecystitis, urinary tract infections, blood disease, injuries, dysentery, edema, influenza, and other diseases [48].
"Sman-dpyad Zla-bavirgyal-po" divides "Dida" into three categories according to its origin: "Indian Dida" ("Jadi", ་ཏི ག), "Nepalese Dida" ("Wadi", བལ་ཏི ག), and "Tibet Dida" ("Wodi", བོ ད་ཏི ག) [7]. "Shel Gong Shel Phreng" followed the above classification and further divided "Tibet Dida" into six groups: "Songdi" ( མ་ཏི ག), "Saierdi"(གསེ ར་ཏི ག), "Edi" (ད ལ་ཏི ག), "Sangdi" (ཟངས་ཏི ག), "Jihedi" ( གས་ཏི ག), and "Gouerdi"( ར་ཏི ག) [8]. "Shel Gong Shel Phreng" recorded that "Jiadi is like a shrub and the whole plant is shiny. The stem is hollow and knobbly and the epidermis is thin and hard. The leaves are thick and dark green" [8]. It is widely believed that S. sinensis is the material medica resource of "Jiadi" [49,50]. S. sinensis is mainly distributed in India and Nepal and was recently found to be distributed in China (Dingjie and Jilong conty in Tibet) [51]. "Shel Gong Shel Phreng" documents that "In contrast to Jiadi, the color of Wadi is light and soft and the leaves are slightly yellow, whereas the other characteristics are similar" [8]. According to literature records and textual research, S. ciliate, S. racemosa, and C. pedunculatum are the material medica resources of "Wadi", among which, the morphological characteristics of S. ciliate are more consistent with the picture of "Wadi" in Mantang ( ན་ཐང་ ལ་འ ེ ལ།, Figure 2) [52]. "Tibetan medicine" considers that C. pedunculatum was used as "Wadi" in some regions; however, more evidence is needed to confirm these findings [5]. "Shel Gong Shel Phreng" documented that "Songdi grows in rock mountains, grassy slopes, under forests, and rock crevices. The stem is red and the leaves are small. The basal leaves are dense like rosettes with red and yellow flowers" [8]. The literature suggests that the material medica resources of "Songdi" are mainly from Saxifraga [5,8,47]. "Shel Gong Shel Phreng" documented that "Saierdi grows in fields and the beach. The stem and leaf are upward extensions with red or yellow flowers. The seeds are yellow and small" [8]. The material medica resources of "Saierdi" are also from Saxifraga in the literature [5,8,47]. "Shel Gong Shel Phreng" documents that the "stems and leaves of Edi are quite long and the flowers are white" [8]. Currently, the doctors in Tibet mainly use species from Parnassia as "Edi", whereas those in Qinghai mainly use species in Cerastium as "Edi" [5,47]. However, their morphological characteristics are not consistent with the above description. The material medica resources of "Edi" remain to be further characterized. According to "Shel Gong Shel Phreng", "Sangdi" is characterized by "tufted and red stems, clustered leaves, and slightly hairy, red and yellow flowers" [8]. This is similar to the genus Saxifraga, but according to the actual use situation in various regions, "Sangdi" is considered a variant of Swertia, which gradually formed in the long history of use under the influence of the distribution of resource species in different regions [5,47]. As recorded in "Shel Gong Shel Phreng", the stems of "Jihedi" are like iron chopsticks; the leaves are green, the flowers are light blue or blue, and the fruits are similar to flax seeds [8]. The literature holds that the material medica resources of "Jihedi" are mainly species of Halenia and Gentiuanopsis [5,47,49,53]. Other Swertia and Lomatogonium plants recorded in Tibetan Medicine as "Jihedi" may be regional substitutes. "Shel Gong Shel Phreng" records that "Gouerdi grows in the shady area of green slopes. The leaves are thick, basal, and with beady, silver spots. The flowers are pale-yellow" [8].
These likely appear to be plants of Saxifraga or Parnassia [5]. However, some literature considers S. nigroglandulifera and Mentha haplocalyx as "Gouerdi", which need to be further confirmed [47,54].

Others
Many other Gentianaceae plants are also used in Tibetan medicine. "Ganggaqiong" (གང་ག་ ང་།), "Shel Gong Shel Phreng" recorded that "It tastes bitter, has heat-clearing and detoxifying effects, and cures blood diseases and Chiba disease. It grows in mountains at high altitudes and the roots are similar to tendons. The plants are fluffy and messy with overlapping leaves, which are similar to eight-corner pagodas. The flowers are in white and urceolate in shape" [8]. Doctors in Tibet and Qinghai (Yushu) mainly use G. urnula as "Ganggaqiong" [5]. Furthermore, in some regions of Tibet, G. phyllocalyx was used as "Ganggaqiong·Manba" (གང་ག་ ང་དམན་བ།) [47]. "Shel Gong Shel Phreng" documents that "Saibao·Guzhui (སེ ར་པོ ་ ་ ས།) can detoxify and cure sores. It can be divided into two types and the top grade grows in the mountains. The stems are very long and the leaves are thin and smooth similar to those of Jieji·Gabao. The flowers are pale yellow" [8]. Doctors in Tibet mainly use S. multicaulis and S. kingie as "Saibao·Guzhi" [47].

Formulas
The clinical application of Tibetan medicine is mainly as formulas and single herb preparations are very few. Formulas can contain more than 100 drugs, and those with more than 10 drugs are usually common. Similar to traditional Chinese medicine formulas, Tibetan medicine formulas also employ the "Monarch, Minister, Assistant, and Guide" methods. Some drugs must be processed before use to eliminate and reduce their toxicity and improve efficacy. Decoctions, powders, and water pills are the most common dosage forms, of which powders and water pills are the most widely used in a clinical setting [4,55].
According to our statistics, about 56 formulas containing "Jieji", 53 formulas containing "Bangjian", 160 formulas containing "Dida", and 14 formulas containing "Gang-gaqiong" were used (Supplement Table S1). "Jieji" has mostly been used in formulas owing to its effect of clearing heat toxins, removing food stagnation, dispelling wind, and eliminating dampness, and purging the liver and gallbladder. "Bangjian" has mostly been used in formulas owing to its effect of clearing lung heat. "Dida" has been mostly used in formulas because of its heat-toxin clearing effect and purging the liver and gallbladder. "Ganggaqiong" has mostly been used in formulas owing to its effect of clearing heat toxins.

Phytochemistry of Gentianeae
To date, most phytochemical studies have mainly focused on the Gentiana and Swertia genera. Nearly 600 metabolites were identified from the Gentiana genus [56][57][58] and more than 400 metabolites were identified from the Swertia genus [59][60][61][62]. The primary bioactive components isolated from the two genera include iridoids, xanthones, flavonoids, and triterpenoids, of which a few are alkaloids, lignans, and phenolic compounds.

Iridoids
As the main chemical constituents, iridoids are widely distributed within the Gentiana and Swertia genera [16]. Iridoids can be divided into six groups including loganic acid, secologanic acid, morroniside, sweroside, swertiamarin, and gentiopicroside derivatives based on the classic mevalonic acid pathway [63]. Loganic acid derivatives mostly belong to carbocyclic iridoids and most are esters of loganic acid [63]. To date, more than 40 loganic acid derivatives were isolated from Gentiana [56], whereas only 10 were isolated from Swertia [61]. Secologanic acid derivatives are mainly derived from the split C7-C8 bond of carbocyclic iridoids. About 19 secologanic acid derivatives were obtained from Gentiana [56,63] and only two (vogelioside and nervoside) from Swertia [16]. Morroniside derivatives were not the major iridoids in both genera. To date, only 13 morroniside derivatives were isolated from Gentiana and none from Swertia [64]. Sweroside, swertiamarin, and gentiopicroside derivatives were not only the major compounds in Gentiana and Swertia but are also widely distributed [16,56,62]. In the biosynthetic pathway, sweroside, swertiamarin, and gentiopicroside are the precursors of the latter [63]. Gentiopicroside derivatives were characterized in the existence of double bond in C-5 and C-6, whereas sweroside derivatives without the double bond and swertiamarin derivatives with hydroxyl at C-5, 22, 14, and 25 of sweroside, swertiamarin, and gentiopicroside derivatives was obtained, respectively, in Gentiana while were obtained respectively in Swertia [56,61].

Pharmacology of Gentianeae
The bioactivities of plants from Gentianeae include hepatic protection, upper respiratory tract protection, joint and bone protection, glucose regulation, antibacterial, antioxidant, anticancer, and antiviral effects ( Table 2).

Hepatic Protection
"Dida" is the most common medicinal material for the treatment of hepatitis, especially icteric hepatitis [48]. The mechanism mainly involves restoring abnormal physiological characteristics and biochemical indexes of liver, improving antioxidants levels and lipid peroxidation, and promoting blood supply to the liver tissue [80][81][82][83]. Plants from "Jieji" also showed similar hepatoprotective effects [84]. Gentiopicroside and Swertiamarin, the main iridoid in "Jieji" plants, can significantly reduce the abnormal biochemical indexes of liver and increase the antioxidants and lipid peroxidation levels [85][86][87].

Protection from Upper Respiratory Tract Infections
"Bangjian" is widely used in the treatment of upper respiratory tract infections. More than 60% of the formulas with "Bangjian" as the primary drug are clinically used for the treatment of respiratory diseases such as bronchitis, emphysema, acute and chronic pharyngitis, cough and asthma, and hoarseness (Table S1). Pharmacological studies reveal that the mechanism involves enhancing p-ERK expression [88] and antioxidant enzymes activities [89], while inhibiting TNF-α, IL-10 and TGF-β1 expression [88,90,91].

Joint and Bone Protection
"Jieji" can effectively protect joints and bones. The extracts of G. straminea, G. macrophylla and Sinogentiana striata were proved to alleviate adjuvant arthritis, synovial inflammation and rheumatoid arthritis by effect on different targets and pathways [92][93][94]. Gentiopicroside and swertiamarin also played an important role in joint and bone protection through inactivation of JNK and NF-кB signaling pathways or interfere with the release of inflammatory factors [95][96][97].

Glucose Regulation
Many compounds from Gentianeae, especially xanthones and iridoid derivatives, were reported to have glucose-regulating effects [98][99][100]. Demethylbellidifolin can stimulate the GLP-1 receptor in a concentration-dependent manner and reduce blood glucose levels [101]. Bellidifolin was observed to reduce blood glucose levels, indicating its hypoglycemic effect [102]. Swertiamarin can significantly reduce the levels of fasting blood glucose, HbA1c, TC, tTG, and LDL; increase the levels of hemoglobin, insulin, TP, and HDL; and significantly promote the regeneration of pancreatic islet tissue in diabetic rats [103]. Swertiamarin may regulate the expression of related genes in the liver and adipose tissue by targeting PPARγ [104].

Antioxidant Effects
The extract of S. chirata, S. davidii and G. urnula was found to have the high freeradical scavenging capacity, which is associated with phenolic compounds and synergistic effects with xanthones and glycosides [109]. Xanthones are the major compounds in Swertia and this species may, therefore, prove useful in scavenging OH·and O 2 . Studies report an increase in radical-scavenging activity when the substituents are present in the orthoposition [110].

Antiviral Effects
Both aqueous and alcohol extracts of G. macropylana significantly inhibit influenza A and B [121,122]. The extracts of G. veitchiorum showed obvious inhibitory effects on RSV both in vivo and in vitro [123]. Lots of compounds isolated from Gentianeae had significantly anti-HBV and anti-HIV activity [79,[124][125][126][127].

Limitation
This review paper is based on the author's own analysis and summary of the literature. Although the author tries to keep objective in the analysis process, it is still highly subjective, thus all the findings are based on personal views. This review paper only covers the research results published in mainstream journals from 2000 to 2021, and it is inevitable to overlook some of them. Therefore, readers need to understand the limitations of this review paper in terms of time and sources. There have been previous reviews on the chemical constituents and pharmacological activities of Gentiana and Swertia [56,60]. Nevertheless, in this review, we mainly focused on the related studies of Gentianeae plants used in Tibetan medicine, especially ethnomedicinal usage. Moreover, we also included the latest research results.

Conclusions
Due to its complete theoretical system and remarkable therapeutic effect, Tibetan medicine has attracted increasing attention from researchers. However, proper research on Tibetan medicine is scarce. In this study, we employ Gentianeae as a case, aiming to provide good inspiration for the follow-up research in Tibetan medicine.
Species from Gentianeae have been widely and long used as Tibetan medicine. Many species are good sources for chemical and pharmaceutical research owing to the presence of high levels of iridoids, flavonoids, and triterpenoids. However, the ethnomedicinal usage and the phytochemical and pharmacological properties of Gentianeae in Tibetan medicine were not well summarized. In view of this, we systematically summarize the ethnomedicinal usage and the phytochemical and pharmacological properties of Gentianeae in Tibetan medicine.
Although various classes of compounds were identified and their pharmacological activities investigated, systematic studies are lacking for numerous species. Thus, there is the likelihood of the presence of undiscovered compounds. Therefore, phytochemical profiling, bioactivity screening, biosynthetic pathway elucidation, and structure-activity relationship studies should be continued, as these findings could provide a more reasonable foundation for use of Gentianeae species and the maximization of their desired therapeutic benefits.