Natural Products, Traditional Uses and Pharmacological Activities of the Genus Biebersteinia (Biebersteiniaceae)

Medicinal plants have been known as a rich source of natural products (NPs). Due to their diverse chemical structures and remarkable pharmacological activities, NPs are regarded as important repertoires for drug discovery and development. Biebersteinia plant species belong to the Biebersteiniaceae family, and have been used in folk medicines in China and Iran for ages. However, the chemical properties, bioactivities and modes of action of the NPs produced by medicinal Biebersteinia species are poorly understood despite the fact that there are only four known Biebersteinia species worldwide. Here, we reviewed the chemical classifications and diversity of the various NPs found in the four known Biebersteinia species. We found that the major chemical categories in these plants include flavonoids, alkaloids, phenylpropanoids, terpenoids, essential oils and fatty acids. We also discussed the anti-inflammatory, analgesic, antibacterial, antioxidant, antihypertensive and hypoglycemic effects of the four Biebersteinia species. We believe that the present review will facilitate the exploration of traditional uses and pharmacological properties of Biebersteinia species, extraction of the NPs and elucidation of their molecular mechanisms, as well as the development of novel drugs based on the reported properties and mode-of-action.


Introduction
Biebersteinia is the smallest genus of Biebersteiniaceae. The systemic and taxonomic position of this genus has long been in dispute due to its rare species and limited availability of representative herbarium collections [1]. The genus was traditionally positioned in Geraniales 30 years ago, but now it was accepted that it belongs to Sapindales as a separate order and family based on the molecular
The content of total flavonoids (CTF) in plants may be correlated with their habitats, ecological roles and responses to abiotic and/or biotic stresses [17][18][19]. In general, the Biebersteinia species are widely distributed at high elevations, and are exposed to extreme drought, low temperatures and strong ultraviolet radiation [20]. All of these conditions could induce high production of CTF. One of our previous studies showed that the CTF reached 0.24% in B. heterostemon located on the Qinghai-Tibetan Plateau [21]. CTF may also widely vary among different plant organs and tissues. For instance, in B. multifida, leaves were found to have the highest CTF (39.9 ± 2.1 mg/g), followed by flowers, stems and roots [22].

Phenylpropanoids
Three phenylpropanoids have been isolated so far from two Biebersteinia species ( Figure 3B; Table 1). Compounds 33 and 34 are coumarins that were identified in B. multifida, which differ only in terms of their substituents at C-6. Compound 35 is a ferulic acid that was isolated from B. multifida [23], as well as from B. heterostemon by our group (unpublished data).

Applications in Traditional Medicines
Among the four well-known Biebersteinia spp., only B. heterostemon and B. multifida have been commonly applied as traditional herbal medicines to treat musculoskeletal disorders, bone fractures and skin diseases [7,25,55]. In China, B. heterostemon plants are widely distributed in Qinghai-Tibetan Plateau, and have been administered as traditional Tibetan medicines [20,56]. In addition, B. multifida is indigenous to Iran, where this plant species has been topically applied as a folk remedy for treatments of muscle and skeletal disorders and bone fractures [9,57]. Besides, it has also been reported that children's nocturia can be treated with B. multifida [58]. In addition, B. odora has been used in treatments of migraine and fever for centuries by people living in the Shigar Valley, Baltistan region of Karakorum range, Pakistan [8]. As Biebersteinia species have high pharmacological values as traditional medicines, their bioactivities have attracted the attention of a large number of phytochemists and pharmacologists.

Anti-Inflammatory and Analgesic Effects
The anti-inflammatory effect of B. heterostemon has been evaluated with a xylene-induced murine inflammation model. Its analgesic effect on mice was established by the hotplate and tail flick methods and by acetic acid-induced writhing [55]. Traditional B. heterostemon decoctions, traditional B. heterostemon decoctions followed by alcohol precipitation, and ethanolic B. heterostemon extracts inhibited xylene-induced ear edema in mice and elevated the mouse hotplate pain threshold [55]. However, the anti-inflammatory and analgesic efficacies of the ethanolic B. heterostemon extract were significantly stronger than those of the other afore-mentioned extracts [55]. These results might be correlated with those for N-3-methyl-2-butenyl urea (40) isolated from the ethanolic extract of B. heterostemon, as this compound was confirmed to have analgesic activity [11]. Similar findings were obtained and reported for B. multifida. A dose of 10 mg/kg B. multifida root extract obtained by ethanol refluxing, and that of 4 mg/kg indomethacin had similar anti-inflammatory efficacies in a carrageenan-induced edema assay [57]. The first phase of a formalin test indicated that the analgesic efficacy of 50 mg/kg B. multifida root extract was comparable to that of 10 mg/kg morphine [57]. These findings collectively indicate the high potential of B. heterostemon and B. multifida for the production of anti-inflammatory and analgesic drugs.

Anti-Fatigue and Anxiolytic Effects
The anti-fatigue effect of B. multifida root extract was also validated in a forced swimming test (FST), and the biochemical parameters in the blood related to fatigue were measured [7]. The results demonstrated the potential benefit of B. multifida root extract as an anti-fatigue material, and showed that it improved physical stamina [7]. These properties and effects might account for the fact that B. multifida has been used in Iranian folk medicine to enhance physical strength [10]. In addition, B. multifida total root extracts exhibited anxiolytic effect in an elevated plus-maze assay [23]. This finding led to the isolation and characterization of three active coumarin derivatives from B. multifida root extracts, namely umbelliferone (33), scopoletin (34) and ferulic acid (35) with the well-known potent monoamine oxidase (MAO) inhibitory and anti-anxiety effects [23,65,66]. These discoveries explain and provide scientific evidence to support the traditional use of B. multifida for the management of anxiety.

Hypolipidemic Effect
It has been well-established that lipoproteins play vital roles in atherosclerosis [67,68]. Over the past several decades, a number of studies have indicated that low-density lipoproteins (LDL) and high-density lipoproteins (HDL) have opposite influences as risk factors in the onset and progression of atherosclerosis [67,[69][70][71][72][73]. It was verified in the last 20 years that lowering LDL-cholesterol successfully prevents atherosclerosis [74]. B. multifida root extracts, prepared by using a solution of water and ethanol with the ratio of 1:2, significantly reduced both the HDL and LDL levels in mice serum at doses of 4 and 5 mg/kg, respectively [75]. In addition, the hydro-methanolic extract of B. multifida roots was recently observed to possess a protective effect on ethanol-induced gastric ulcer in rats, which was thought to be partly related to antioxidant activity and accelerating nitric oxide (NO) production in vivo after the rats were treated with the extracts [76]. Taken together, Biebersteinia species can be explored for a wide range of pharmacological activities.

Antimicrobial Effects
A number of studies have reported that B. heterostemon and B. multifida extracts significantly inhibited the growth of various bacteria and fungi in a concentration-dependent manner. For instance, the whole plant extracts of B. heterostemon substantially inhibited the growth and proliferation of the pathogenic fungi Fusarium equiseti, F. oxysporum and F. moniliforme, which are thought to be the causes of inducing the Chinese Angelica stem nematode disease, with the minimum inhibitory concentrations (MICs) of 0.6250 mg/mL, 0.6250 mg/mL and 1.2500 mg/mL, respectively [77]. Another independent study estimated the antibacterial activities of root extracts from B. multifida against various Gram-positive or negative bacteria, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes and Salmonella enterica [78]. The results unraveled that the root extracts of B. multifida obtained by n-hexane and ethanol maceration displayed significant antibacterial effects with the MIC of 0.195 mg/mL [78]. Some terpene compounds isolated from B. heterostemon were confirmed to possess antibacterial activities. Moreover, the compound (-)-anymol-8-O-β-D-lyxopyranoside (38), a bisabolane-type sesquiterpene glycoside isolated from B. heterostemon, displayed a pronounced antibacterial efficacy against B. subtilis, S. aureus and Pseudomonas spp. with the MICs of 50 µg/mL, 50 µg/mL and 70 µg/mL, respectively [26]. In addition, the prenylated guanidine known as galegine (30) was reported to exhibit the most potent antibacterial efficacy against various S. aureus strains, including the two methicillin-resistant ones, in the concentration range between 20 and 31 µM [79,80].

Antioxidant Activities
Numerous studies have reported on the antioxidant activities of dietary phenolic substances like flavonoids in various living organisms, including plants, animals and humans [81][82][83][84][85][86]. Most of the published reports have focused on the antioxidant activities of phenolics possessing the ability to inhibit the formation of free radicals, the mode of which depends mainly on the structure-activity relationships of antioxidant compounds [87][88][89][90]. High CTF in Biebersteinia plants is closely correlated with their antioxidant activities [17]. Various methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging approaches, and Oil Stability Index (OSI) assay, have been used to determine the antioxidant activities of different B. heterostemon solvent extracts, and disclosed that their antioxidant activities varied considerably [91]. Specifically, the ethyl acetate and ethanolic extracts of B. heterostemon aerial parts were presented with higher antioxidant activities than the n-hexane extract, and their relative efficacies were concentration-dependent [91]. One possible explanation is that flavonoids and phenols were more readily extracted with polar than nonpolar solvents [92][93][94]. In addition, a B. multifida root extract was found to be enriched with phenolic compounds (80.1 ± 3.10 mg/mL), and demonstrated strong DPPH radical-scavenging activity (95.9 ± 3.20 µg/mL) [21]. It is worth mentioning that the polyphenolic compounds identified in food products prepared from various plant sources like Avena sativa, Aristotelia chilensis, Paeonia ostii and Linum usitatissimum possess significant antioxidant activities as well [95][96][97][98].
Besides antioxidant activities related to phenolic compounds present in Biebersteinia spp., the essential oils from different types of tissues of Biebersteinia plants were also shown to have strong radical-scavenging activities. For instance, the essential oil of B. multifida fruits, evaluated by DPPH assay, was shown to be superior to essential oils extracted from other organs (e.g., leaves and roots), displaying the IC 50 value of 16.7 ± 0.02 µg/mL that was even more excellent than the well-known synthetic antioxidant butylated hydroxytoluene (BHT, 19.0 ± 0.80 µg/mL) [45]. The chemical composition in the essential oils of B. multifida fruits should be responsible for their antioxidant activity due to the antioxidative properties of thymol, 1,8-cineol and β-caryophyllene [45], which are the major compounds in B. multifida essential oils [99,100]. These investigations indicate that the Biebersteinia species are a valuable natural resource for extracting antioxidant compounds.

Anti-Cancer Effects
A growing body of literature has demonstrated that Biebersteinia species possess some other valuable pharmacological effects, in addition to those overviewed in previous subsections. For instance, an ethanolic extract from B. multifida roots was reported to prevent mutation reversion by 51.2% in an anti-mutagenicity test, indicating that B. multifida plants harbor natural products that can act as anticancer agents [101]. Another independent study showed that the root extract of B. multifida obtained by maceration with 70% ethanol was cytotoxic and apoptotic to both human prostate cancer cells DU145 and PC3 in a dose-dependent manner, as this extract significantly decreased cell viability [102].

Conclusions and Future Perspectives
Natural plant products have been used extensively and widely in traditional medicine, and are important sources for drug discovery and development. Up-to-date, only a few studies have examined and analyzed the phytochemical constituents, bioactivities, and pharmacological aspects and characteristics of Biebersteinia species. More than 40 secondary metabolites have been isolated and identified in the members of this plant genus, of which flavonoids were the principal constituents. The varied properties and efficacies of the pharmacologically active substances in different Biebersteinia species suggest that these compounds are potential sources of new drugs.
However, certain key issues must be resolved before the identified Biebersteinia species can be fully exploited as bases for new pharmaceutical agents. Currently, many of their phytochemical constituents have not yet been systematically identified, and some of those that have already been elucidated do not necessarily account for their observed pharmacological effects. Although certain constituents have significant pharmaceutical effects, their underlying mode-of-action and molecular mechanisms remain unclear. Moreover, in vivo and in vitro models should be designed and implemented in order to screen for unrecognized bioactivities. For instance, although B. heterostemon is widely used in folk medicine in northwest China, it has also generally been regarded as toxic, or a weed that is difficult to be eradicated. Consequently, the potential utility of this resource has been underexploited, or even was lost altogether. In order to harness the full value of the identified Biebersteinia species as pharmaceutical agents, we should perform basic research on their bioactive constituents, pharmacological properties and molecular mechanisms, which is followed by clinical tests. In-depth investigations are therefore required to develop, test and optimize the administration of novel drugs derived from various organs of plants of this genus. Overall, we believe that this synopsis will facilitate the development and exploitation of new drug resources from these plant materials.
Author Contributions: All authors listed have made substantial direct and intellectual contributions to the work, and approved it for publication. All authors have read and agreed to the published version of this manuscript.

Conflicts of Interest:
The authors declare no conflicts of interest.