Advances in chemistry and bioactivity of the genus Erythroxylum

Erythroxylum P. Browne is the largest and most representative genus of Erythroxylaceae family. It contains approximately 230 species that are mainly distributed in tropical and subtropical regions. Some species in this genus, such as E. monogynum and E. coca, have been used as folk medicines in India or South America for a long history. It is well known that Erythroxylum plants are rich in tropane alkaloids, and the representative member cocaine shows remarkable activity in human central nervous system. However, many other types of active compounds have also been found in Erythroxylum along with the broadening and deepening of phytochemical research. To date, a total of 383 compounds from Erythroxylum have been reported, among which only 186 tropane alkaloids have been reviewed in 2010. In this review, we summarized all remained 197 compounds characterized from 53 Erythroxylum species from 1960 to 2021, which include diterpenes, triterpenes, alkaloids, flavonoids, and other derivates, providing a comprehensive overview of phytoconstituents profile of Erythroxylum plants. In addition, the biological activities of representative phytochemicals and crude extracts were also highlighted. Graphical Abstract


Introduction
In the long evolutionary process of nature, plants have acquired the ability to synthesize various compounds to better adapt to stimulations in the environment. The accumulation of practical experience has made human realize that these substances are also of significant importance for the treatment of human diseases and

Open Access
Natural Products and Bioprospecting the improvement of the quality of life. With the support of technology in compound extraction, separation and structural identification, the active substances in traditional herbs are gradually being discovered by humans. Therefore, modern medicine based on a single or several compounds has been developed. With the deepening of research on plant natural products, new biologically active compounds are constantly being discovered and further applied in medicine, health care and agriculture. Sorting out and summarizing the plant distribution, structure, and activity characteristics of these newly discovered phytocompounds will confer us effective information in rational use of plant resources.
Erythroxylum P. Browne, the representative genus of Erythroxylaceae family, is especially well known for its phytoconstituents of tropane alkaloids (TAs), such as cocaine [1,2]. Species of this genus are mainly distributed in tropical and subtropical regions including South America, South Africa, Southeast Asia and Australian flora [2]. As the largest genus of the Erythroxylaceae family, approximately 230 species are included in Erythroxylum [2], among which E. coca and E. novogranatense are the famous plant sources of cocaine. Before achieving the purification of cocaine from plants in 1859 [3], the leaves of E. coca or E. novogranatense had been chewed by the Indigenous South American as stimulant and hungersuppressant for over a thousand years. The remarkable biological activity of cocaine in human central nervous system attracted widespread attention to compounds in plants of this genus. Accordingly, numerous of cocaine analogs (TAs), as well as other bioactive compounds have been found in Erythroxylum [4,5].
To date, no comprehensive summary on chemical compositions found in Erythroxylum species and their bioactivities has been reported, though Oliveira et al. [6] presented an excellent review focusing on structures of TAs isolated from this genus in 2010 and Dr. John D' Auria' s group discussed application potentials of Erythroxylum species worldwide in mental health, nutrition, agriculture, and commercialization based on studies on representative compounds discovered in this genus [7]. Attracted by the diverse biological activity of compounds found in Erythroxylum, which included anaesthetic [8], antioxidative [9,10], anti-inflammatory [9], cytotoxic [11], anticancer [12], and insecticidal activities [13], as well as neutralization of snake venom [14], we therefore aimed to provide a comprehensive review of all compounds reported in Erythroxylum species from 1960 to 2021 and an update of alkaloids isolated after 2010 here, which is supposed to be essential for further effective development and utilization of plant resources in the genus in the future. Additionally, we also presented an overview of the biological activities of representative phytochemicals and crude extracts at the end of the review, providing medicinal and commercial application prospects of Erythroxylum species.

Chemical composition
Based on the published results dedicated to study chemical composition of Erythroxylum species, 383 compounds, including diterpenes, triterpenes, flavonoids, alkaloids, and other derivates, have been found in 67 Erythroxylum species. Among these, 186 TAs compounds identified in Erythroxylum plants before 2010 have been systematically reviewed by Oliveira et al. [6]. Therefore, here we summarized all remained 197 compounds characterized from 53 Erythroxylum species from 1960 to 2021, which include diterpenes, triterpenes, alkaloids, flavonoids, and other derivates.
According to the literature, Erythroxylum plants are rich in alkaloids. Especially E. coca, E. coca var. coca, and E. novogranatense var. novogranatense, the content of total alkaloids varies from 0.5% to 2.4% in leaves (dry mass, Table 1) [15]. Particularly, high cocaine content (0.13%-0.76% dry mass) was found in E. coca and E. novogranatense leaves [16]. In 2006, Stefan Bieri et al.  [17] analyzed the cocaine distribution in 51 plant species and cocaine was detected only in 23 Erythroxylum species with the content less than 0.001% (dry leaves). High production of total phenols, total tannins and total flavonoids of up to 17.97%, 8.4%, and 3.87% (dry leaves), respectively, was reported in E. suberosum, E. tortuosum, and E. deciduum [18] (Table 1). Additionally, the total diterpenes content determined in stems of E. australe and E. pictum ranged from 0.09% to 1.8% (dry mass, Table 1).

Diterpenes
Plants of Erythroxylum are rich in diterpenoids, which have been extensively studied since the last century. In particular, Connolly [21][22][23][24] and Kapadi [25][26][27], who focused on investigating diterpenoids of E. monogynum in 1960s, provided the earliest knowledge of diterpenoids in Erythroxylum species. Using nuclear magnetic resonance (NMR) spectroscopy and chemical reactions, they and their coworkers elucidated the structures of 17 diterpenoids in E. monogynum. To date, about 11 types of diterpene skeletons (a-k) have been identified from plants in this genus (Fig. 1). Based on the number of rings in the diterpene skeletons, diterpenes found in Erythroxylum species could be divided into bicyclic diterpenes, tricyclic diterpenes, and tetracyclic diterpenes.

Flavonoids
Flavonoids are a large and complex group of constituents found in almost all plants. Flavonoid variation in thirteen species of Erythroxylum has been studied systematically by Plowman et al. in 1988 [59]. They found kaempferol, ombuin (7,4ʹ-dimethylquercetin), and quercetin were predominant flavonoid aglycones in Erythroxylum plants analyzed. Besides, Johnson and coworkers [60][61][62][63][64][65], based on their work on flavonoids profiles of six species or variants and flavonoids that had been reported in Erythroxylum, proposed that some unique flavonoids could be used as chemotaxonomic markers for taxon. Overall, flavonoid aglycones in Erythroxylum mainly consist of quercetin, ombuin, fisetin, kaempferol, epicatechin, eriodictyol and taxifolin. In addition to these, isoflavone, isoflavanone and other flavone derivatives were also found in Erythroxylum. Chemical structures of flavonoid aglycones that have been found in Erythroxylum plants were summarized and presented in Fig. 7. Moreover, the major glycosides of these flavonoids include mono-glucosylrhamnosyls and dirhamnosyl-glucosides, as well as mono-galactosyl and mono-arabinosyl. In total, 73 flavonoids from 37 species of Erythroxylum have been studied (Table 7), though some structures lack NMR data support in the literature.
Phenolic derivatives and their glycosides were also obtained ( Fig. 8;    showed significant anti-oxidant and anti-glycation activities in vitro [77].

Bioactivities of diterpenes, triterpenes and sterols
Pharmacological investigation of diterpenes isolated from Erythroxylum species are still scarce despite the large resource found. Diterpene 14-O-methyl-ryanodanol (77) showed insecticidal activity against Aedes aegypti larvae [13], as well as a dose-dependent cytotoxic effect to astrocytes (GL-15 cell line) [78]. Cytotoxicity activities against five tumor cell lines of devadarane derivatives (69, 73-75) were also investigated, but no activity was observed [35]. Exploring and evaluating bioactivities of the numerous diterpenoids found in Erythroxylum species will be essential for further effective utilization of these natural product resources in this genus. For triterpenes, compounds 93-95 were major constituents of the hexane extract of E. daphnites leaves which showed a cytotoxic effect against SCC-9 oral squamous cell carcinoma cell line [48]. Additionally, sterols (193, 194) isolated from E. monogynum possess good anti-oxidant and anti-glycation activities [77]. Additionally, although a large number of flavonoids have been found in Erythroxylum species, these compounds are not specifically distributed in this genus. Readers interested in the details of bioactive flavonoids are referred to the review by Shashank Kumar and coworkers [79].

Bioactivities of TAs
In Erythroxylaceae family, TAs specially occur in species of Erythroxylum. Until now, a total of 197 TAs compounds have been characterized in Erythroxylum plants.

Bioactivities of crude extracts
In addition to research on single compound, many studies have been carried out on the biological activities of crude extracts of Erythroxylum plants (Table 9). E. monogynum is rich in alkaloids and diterpenes. In 2019, Dhanunjaya et al. [95] summarized that crude extracts of this species had multiple bioactivities, such as antioxidant, antihyperlipidemic, antidiabetic, antiplasmodial and hepatoprotective. Particularly, leaf and bark extracts of E. delagoense, E. emarginatum, or E. pictum, showed great antibacterial activities [96]. Ethanolic extract obtained from the roots of E. pungens could induce dose-dependent hypotension and tachycardia in conscious rats, as well as vasorelaxation in mesenteric artery ring preparations in vitro [50]. Ethanolic extract of E. caatingae has a relaxant effect on ovine cervical contractions [97]. Besides, low-polarity fractions of this species showed significantly high cytotoxicity activity against the NCI-H292, HEp-2 and K562 cell lines [12]. Furthermore, acetone/water (70/30, v/v) extract of E. macrocarpum is a significant inhibitor of acetylcholinesterase [98]. Hydroalcoholic extracts of E. areolatum or E. confusum showed antiherpetic activity [99]. For the antitumor activity, when mice were treated with different doses of methanol extract of E. caatingae, a significant reduction in their tumor weight was observed [53]. Moreover, extracts of E. minutifolium or E. confusum showed hepatoprotective effects [100]. Crude extracts, fractions, or isolated products of E. ovalifolium or E. subsessile were demonstrated to inhibit toxic effects of the snake (Lachesis muta) venom, providing a new strategy for antivenom treatment [14].

Conclusions and prospecting
Based on the current progress in phytochemistry of the  triterpenoids obtained from 8 Erythroxylum species, 11 TAs found in 6 species after 2010, 73 flavonoids from 37 Erythroxylum species, and 17 other constituents (norisoprenoids, phenolic derivatives and their glycosides, and steroids). Among these compounds, most diterpenes were isolated from the timber or roots of the plants, triterpenes were identified from aerial organs, flavonoids were distributed in leaves or branches, while others had no obvious tissue-or organ-specific distributions. Significant biological activities, including anaesthetic [8], antioxidative [9,10], anti-inflammatory [9], cytotoxic [11], anticancer [12], and insecticidal activities [13], as well as neutralization of snake venom [14], have been demonstrated for isolated products or crude extracts from some species of Erythroxylum. However, potential activity of most compounds is still unknown. In-depth biological activity studies on compounds obtained will be the basis for exploring potential medicinal resources in this genus. Additionally, some of the diterpenes were suggested to serve as the defensive components to protect the Erythroxylum plants from herbivores, pathogens, or other environmental challenges. Therefore, they could be used as potential bioinsecticides in agriculture in the future. Elucidation of natural product biosynthetic pathways has been proved to be highly useful for natural products discovery, structure identification and subsequent heterologous synthesis. In Erythroxylum plants, TAs and diterpenes are representative phytoconstituents. Biochemists and molecular biologists have long sought to identify the biosynthetic pathways of TAs, especially cocaine, through isotope labeled precursor feeding studies and gene cloning and characterization [105][106][107][108][109][110]. As a result, incomplete biosynthetic route of cocaine starting from arginine and ornithine and passing through putrescine, methylecgonone, and methylecgonine has been established [7,110] (Fig. 9), though further studies are still essential to elucidate the missing steps. Studies focusing on the biosynthesis pathway of diterpenes in Erythroxylum plants have not been reported till now. However, the kaurenetype (Fig. 1i) diterpene synthase that is responsible for the formation of ent-kaurene, the universal biosynthetic intermediate of gibberellin, has been identified in many other plants [111][112][113]. Besides, ent-beyerene synthase, which is the key diterpene cyclase required for generating ent-beyerene type diterpenes (Fig. 1h), has been characterized in monocotyledonous rice (Oryza sativa L.) [114]. Still, much more researches needed to be done for better understanding the biosynthetic mechanisms and diversity of diterpenes identified in Erythroxylum.