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.

1. 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 phylogenetic analysis [2,3]. The genus Biebersteinia was originally recognized to comprise five species—namely, B. heterostemon Maxim., B. multifida DC., B. leiosepala Jaub. & Spach, B. odora Stephan ex Fisch. and B. orphanidis Boiss. decades ago [1]. However, B. leiosepala is now recognized to be a synonymous species of B. multifida (http://www.theplantlist.org/, http://www.worldfloraonline.org/, and https://www.gbif.org/); and thus, there are a total of four species in the genus. These species are widely distributed in mountainous, semi-arid regions from central and western Asia to the eastern Mediterranean [1,2,4,5,6].

All of the Biebersteinia species are perennial herbs, and possess slightly different biological characteristics and geographical distributions. B. heterostemon, also called “Xun Dao Niu” in Chinese, is endemic to the Qinghai-Tibetan Plateau and its adjacent regions in China [6]. This plant species inhabits arid and semi-arid alpine deserts, rocky slopes and other environments (http://www.iplant.cn/foc/). The morphological and biological characteristics of B. heterostemon are 40–120 cm tall, lanceolate leaf blade bearing long simple hairs and small stipitate glands, flowers in two or three fascicles with hairy or glandular pedicel, as well as yellow and obovate petals (http://www.iplant.cn/foc/). B. multifida is a common herb known as Adamak in Iran, with 20–70 cm long stem, laciniate leaves, flowers formed in a lax panicle, calyx strengthened in fruit, and yellowish petals slightly shorter than the sepals [7]. B. odora is distributed widely across central Asia (e.g., Kazakhstan, Kyrgyzstan, Pakistan, India, China and Mongolia) and inhabited in alpine meadows and dry rocky and scree slopes. B. odora is 10–30 cm tall, and has pinnatisect leaves and yellow flowers with orange center (1–1.5 cm across, in racemes) [8]. B. orphanidis is the only species distributed in Europe and found in Greece besides Asia. B. orphanidis grows at altitudes ~1400–1750 m in deep sandy-clay soil in dolines over limestone, usually in openings of Abies cephalonica forest. These plants are 15–40 cm long, broadly oblanceolate in outline, with scarious stipules and short petioles [5].

Of these species, B. heterostemon and B. multifida have long histories as traditional folk medicines in Iran and the Tibetan region of China, respectively, and have been used to treat various diseases. Modern pharmacological studies have shown that these two plant species have significant pharmaceutical effects on humans, and possess various ethnomedicinal properties, including antioxidant, analgesic, anti-inflammatory, antispasmodic, hypoglycemic, hypotensive and anti-atherosclerotic properties [9,10,11]. Therefore, numerous phytochemists and pharmacologists worldwide have investigated the pharmacodynamically active substances in Biebersteinia species. Natural products isolated from Biebersteinia plants include flavonoids, guanidines, alkaloids, phenylpropanoids, terpenoids, sterols and fatty acids, as well as various compounds of essential oils. The present review summarizes the findings of several decades of research into the chemical constituents and pharmacological functions of the four identified Biebersteinia species. This review, therefore, will facilitate further investigations into the complete chemical profile of the secondary metabolites in these plants, as well as their pharmacological activities and molecular mechanisms.

2. Data Collections

All data presented in this review were summarized from the references, including scientific journals, book chapters or dissertations. These references were systematically searched against electronic databases: PubMed, CNKI (http://new.oversea.cnki.net/index/), Web of Science, Scopus and Google Scholar with a keyword “Biebersteinia”. To search for maximum relative references, the keyword was set as “Biebersteinia” without any other restrictions. Subsequently, references closely related to chemical compositions, traditional uses and pharmacological properties, including in vitro and in vivo investigations, were screened for further data extraction. In addition, to survey the taxon, phenotypes and geographical distributions of species in Biebersteinia, several online taxonomic databases, including http://theplantlist.org/, http://www.worldfloraonline.org/, https://www.gbif.org/ and http://www.iplant.cn/foc/, were also explored.

3. Natural Products Isolated from Bieberstrinia

3.1. Flavonoids

Up-to-date, 29 flavonoids (Figure 1 and Figure 2; Table 1) have been isolated from four Biebersteinia species, which occupies most of the known chemicals in Biebersteinia species. The flavonoid aglycones comprise mainly flavones or flavonols, such as quercetin, luteolin and apigenin. Fifteen aglycone derivatives have been discovered, among which 12 compounds are flavones (1–12) and three are flavonols (13–15) (Figure 1; Table 1). Fourteen flavonoid glycosides with different types or numbers of glycosyl moiety, including 11 flavone (16–26) and three flavonol glycosides (27–29) were found (Figure 2; Table 1).

Figure 1

Chemical structures of 15 flavonoid aglycones identified in Biebersteinia plants. (A) flavone aglycones; (B) flavonol aglycones.

Figure 2

Chemical structures of 14 flavonoid glycosides identified in Biebersteinia plants. (A) flavone glycosides; (B) flavonol glycosides.

Table 1

Chemical constituents (except essential oil-related compounds) in Biebersteinia species.

No.	Compound Name	Sources	References
Flavonoids
1	luteolin	B. heterostemon	[11,13,15]
		B. multifida
		B. orphanidis
2	6-hydroxyluteolin	B. heterostemon	[13]
3	4′-methoxytricetin	B. heterostemon	[14]
4	5,7,3′-trihydroxy-8,4′,5′-trimethoxyflavone	B. heterostemon	[11]
5	apigenin	B. orphanidis	[15]
6	acacetin	B. orphanidis	[15]
7	5,7,4′-trihydroxy-6,8-dimethoxyflavone	B. orphanidis	[15]
8	nevadensin	B. orphanidis	[15]
9	gardenin B	B. orphanidis	[15]
10	acerosin	B. orphanidis	[15]
11	sudachitin	B. orphanidis	[15]
12	hymenoxin	B. orphanidis	[15]
13	quercetin	B. heterostemon	[12]
14	myricetin	B. odora	[16]
15	artemetin	B. heterostemon	[23]
16	hypolaetin-7-O-β-D-xylopyranoside	B. heterostemon	[12]
17	hypolaetin-7-O-β-D-glucopyranoside	B. heterostemon	[13]
18	3′,4′,5,8-tetrahydroxyflavanone-7-O-β-glucopyranoside	B. heterostemon	[24]
19	luteolin-7-O-glucoside	B. heterostemon	[11,13,15]
		B. multifida
		B. orphanidis
20	apigenin-7-O-glucoside	B. orphanidis	[15]
21	tricetin-7-O-glucoside	B. orphanidis	[15]
22	diosmin	B. heterostemon	[13]
23	apigenin-7-O-rutinoside	B. heterostemon	[13,15]
		B. orphanidis
24	luteolin-7-O-rutinoside	B. heterostemon	[13,15]
		B. multifida
25	apigenin-7-O-sophoroside	B. heterostemon	[13]
26	chrysoeriol-7-O-sophoroside	B. heterostemon	[13]
27	quercetin-7-O-glucoside	B. heterostemon	[11]
28	quercetin-3-O-β-glucopyranoside	B. heterostemon	[13]
29	quercetin-3-O-β-D-glucopyranosyl	B. heterostemon	[13]
	(1→2)-β-D-glucopyranoside
Guanidines
30	galegine	B. heterostemon	[12,25]
31	cis-4-hydroxy galegine	B. heterostemon	[25]
32	trans-4-hydroxy galegine	B. heterostemon	[25]
Phenylpropanoids
33	umbelliferone	B. multifida	[23]
34	scopoletin	B. multifida	[23]
35	ferulic acid	B. multifida	[23]
Terpenoids
36	geniposide	B. heterostemon	[26]
37	6β-hydroxy geniposide	B. heterostemon	[26]
38	(-)-anymol-8-O-β-D-lyxopyranoside	B. heterostemon	[26]
Other Types
39	(+)-dehydrovomifoliol	B. heterostemon	[24]
40	N-3-methyl-2-butenyl urea	B. heterostemon	[11]
41	vasicinone	B. multifida	[27]
42	alternariol	B. heterostemon	[24]
43	mannitol	B. heterostemon	[12]
44	β-sitosterol	B. heterostemon	[11,12,24]
45	daucosterol	B. heterostemon	[12]
46	protocatechuic acid methyl ester	B. heterostemon	[24]
Fatty Acids
47	myristic acid	B. orphanidis	[28]
48	palmitic acid	B. heterostemon	[28,29]
		B. orphanidis
49	stearic acid	B. heterostemon	[28,29]
		B. orphanidis
50	arachidic acid	B. heterostemon	[29]
51	docosanoic acid	B. orphanidis	[28]
52	tetracosanoic acid	B. orphanidis	[28]
53	hexacosanoic acid	B. orphanidis	[28]
54	palmitoleic acid	B. heterostemon	[28,29]
		B. orphanidis
55	oleic acid	B. heterostemon	[28,29]
		B. orphanidis
56	eicosenoic acid	B. heterostemon	[28,29]
		B. orphanidis
57	linoleic acid	B. heterostemon	[28,29]
		B. orphanidis
58	α-linolenic acid	B. heterostemon	[28,29]
		B. orphanidis
59	γ-linolenic acid	B. heterostemon	[29]
60	7,10,13-hexadecatrienoic acid	B. orphanidis	[28]

In addition, most of the flavonoid aglycones and glycosides were highly hydroxy- or methoxy-substituted at C-6, C-8, C-3’, C-4’ and C-5’ in their chemical structures (Figure 1 and Figure 2). Both C-6 and C-8 were substituted by methoxy groups as seen in the chemical structures of compounds 7–12 (Figure 1). To the best of our knowledge, this configuration occurs rarely in nature, which might be correlated with their extreme habitats. From the sources of flavonoids, 18 compounds were isolated from the species B. heterostemon (1–4, 13, 15, 16–19 and 22–29) [11,12,13,14], among which compounds 3 and 18 were recently discovered by our group from the species for the first time [14]. Compounds 1, 19, 23, 5–12 and 20–21 were mainly identified from B. orphanidis [15], while three flavonoids (1, 7 and 12) were found in B. multifida; however, only one compound, namely myricetin (14), was reported from B. odora [16] (Table 1).

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].

3.2. Guanidines

Three rare prenylated guanidines, namely galegine (30), cis-4-hydroxy galegine (31) and trans-4-hydroxy galegine (32) [30,31], have been found in B. heterostemon [25] (Figure 3A; Table 1). The clinical hypoglycemic drug metformin was derived from galegine, which might account for the hypoglycemic efficacy of the traditional galegine-containing Tibetan medicine B. heterostemon. In fact, numerous alkaloids [14], such as coptisonine [32], conophylline [33] and vindogentianine [34], can induce hypoglycemia.

Figure 3

Chemical structures of guanidines (A) phenylpropanoids, (B) terpenoids, (C) and other compounds, (D) identified in Biebersteinia species.

3.3. 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).

3.4. Terpenoids

Until the present, four terpenoids have been isolated from B. heterostemon among the four Biebersteinia species. These identified terpenoids include two iridoid glucosides, i.e., geniposide (36) and 6β-hydroxygeniposide (37), and one sesquiterpene glycoside (-)-anymol-8-O-β-D-lyxopyranoside (38) [26]. They are the main active ingredients [35], and are easily hydrolyzed by β-glucosidase to genipin [36]. In addition, we recently isolated one sesquiterpene (+)-dehydrovomifoliol (39) from B. heterostemon, the identification and characterization of which were also reported for the first time from the genus Biebersteinia (Figure 3C; Table 1).

3.5. Other Compounds

Seven other types of compounds were isolated from B. heterostemon, including N-3-methyl-2-butenylurea (40) [11], alkaloid vasicinone (41) [27], alternariol (42) [24], mannitol (43) [12], β-sitosterol (44) [11,12,24], daucosterol (45) [12], and protocatechuic acid methyl ester (46) [24] (Figure 3D; Table 1). In addition, three neutral polysaccharides were obtained from the roots of B. multifida, namely, glucan-A, glucan-B and glucan-C. Their molecular weights were 4100, 2200 and 1100, respectively [37,38,39,40].

3.6. Fatty Acids

Fatty acids, which are aliphatic monocarboxylic acids, can either be saturated or unsaturated depending on the absence or presence of double bonds [41]. A number of studies have reported the presence of various fatty acids in two out of four Biebersteinia species [28,29]. In particular, a total of 14 fatty acids were identified in the seed oil of B. heterostemon and leaves of B. orphanidis (Figure 4; Table 1). These fatty acids include seven saturated fatty acids (47–53), three monounsaturated fatty acids (54–56), and four polyunsaturated fatty acids (57–60). By using gas chromatography (GC) analysis of fatty acids in seed oil of B. heterostemon, nine fatty acids (48–50 and 54–59) were identified, which accounted for 88.44% of total fatty acid content that mainly consisted of unsaturated fatty acids, such as oleic (55), linoleic (57) and linolenic (58 and 59) acids, while the lower detected part (7.94%) of total fatty acid content contained saturated fatty acids, mainly palmitic (48) and stearic (49) acids [29]. In particular, the content of linoleic acid reached 73.04% [29]. Twelve fatty acids were elucidated in the leaves of B. orphanidis (47–49, 51–58 and 60), among which palmitic, linolenic and linoleic acids were predominant, representing 30.60%, 21.83% and 11.67%, respectively, in total fatty acid content [28].

Figure 4

Chemical structures of fatty acids identified in Biebersteinia species. (A) saturated fatty acids; (B) monounsaturated fatty acids; (C) polyunsaturated fatty acids.

4. Chemical Compositions of Essential Oils in Biebersteinia Species

Up-to-date, 112 chemical constituents have been identified in the essential oils of three Biebersteinia species, namely B. multifida, B. heterostemon and B. orphanidis [42,43,44,45,46,47,48], mainly by using gas chromatography-mass spectrometry (GC-MS) analyses (Table 2). In particular, the chemical compositions of essential oils of B. multifida were more systematically investigated, using different types of tissues, such as leaves, fruits and roots [45], or using different extraction methods, such as hydrodistillation, microwave, solvent and supercritical fluid extraction (SFE) [42,44]. In the essential oil of B. multifida, a total of 88 chemical constituents were identified [45,46,47,48]. The chemodiversity and contents of various compounds in essential oils from different parts of B. multifida differed significantly [45]. Specifically, thymol (16.5% of total essential oil), α-pinene (14.3%), β-pinene (12.4%), β-caryophyllene (11.2%) and 1,8-cineol (10.1%) are the major compounds in essential oil extracted from B. multifida leaves; thymol (38.4%), 1,8-cineol (18.4%), γ-terpinene (11.3%) and β-caryophyllene (9.8%) are the main compounds in essential oil extracted from B. multifida roots; and thymol (30.9%), β-caryophyllene (15.5%), α-pinene (9.4%), β-pinene (8.8%), caryophyllene oxide (8.4%) and limonene (7.5%) are predominant in essential oil extracted from B. multifida fruits [45]. Additionally, different extraction methods were also shown to induce different chemical types or contents in essential oil extracts of B. multifida. For example, the hydrodistillation method enabled the authors to mainly detect (E)-nerolidol (31.45%) and phytol (17.1%); microwave extraction allowed detection of (E)-nerolidol (28.4%), n-heptacosane (17.36%), n-docosane (12.97%) and 6,10,14-trimethyl-2-pentadecanone (10.38%); while solvent extraction detected mainly nonacosane (38.62%), mandenol (17.17%) and n-heptacosane (10.23%) [44]. In addition to the above-mentioned extraction approaches of essential oils, the supercritical fluid extraction (SFE) is a green technology that has been widely used in the past few decades to extract essential oils, nonpolar substances, fatty acids, phytosterols, and other functional and nutraceutical components from natural sources [49,50,51,52,53,54]. Four compounds, namely nerolidol, 6,10,14-trimethyl-2-pentadecanone, hexadecanoic acid and phytol that possess strong antioxidant activities, were the major components in essential oils extracted from aerial parts of B. multifida by both hydrodistillation and SFE methods; however, the yield of these four compounds extracted by SFE (91.74%) was far higher than that by the hydrodistillation method [42].

Table 2

Chemical compositions of essential oils in Biebersteinia species.

No.	Compound Name	Molecular Formula	Retention Indices (RI)	Sources	References
1	α-thujene	C10H16	920	B. multifida	[47]
2	α-pinene	C10H16	939	B. multifida	[44,45,46,48]
				B. heterostemon
3	camphene	C10H16	946	B. multifida	[45,48]
				B. heterostemon
4	sabinene	C10H16	970	B. multifida	[45]
5	β-pinene	C10H16	978	B. multifida	[45,48]
				B. heterostemon
6	6-methyl-5-hepten-2-one	C8H14O	988	B. multifida	[47]
7	myrcene	C10H16	991	B. multifida	[45,48]
				B. heterostemon
8	α-phellandrene	C10H16	1005	B. multifida	[45]
9	α-terpinene	C10H16	1018	B. multifida	[45]
10	p-cymene	C10H14	1025	B. heterostemon	[48]
11	limonene	C10H16	1029	B. multifida	[45,48]
				B. heterostemon
12	1,8-cineole	C10H18O	1033	B. multifida	[45,47,48]
				B. heterostemon
13	trans-β-ocimene	C10H16	1050	B. heterostemon	[48]
14	γ-terpinene	C10H16	1062	B. multifida	[45,48]
				B. heterostemon
15	trans-sabinene hydrate	C10H18O	1064	B. multifida	[44,45]
16	linalool	C10H18O	1099	B. multifida	[44,45,48]
				B. heterostemon
17	nonanal	C9H18O	1102	B. multifida	[45]
18	octyl acetate	C10H20O2	1124	B. multifida	[45]
19	cis-limonene oxide	C10H16O	1131	B. orphanidis	[43]
20	trans-pinocarveol	C10H16O	1140	B. multifida	[45]
21	camphor	C10H16O	1143	B. multifida	[44,47,48]
				B. heterostemon
22	(Z)-3-nonenol	C9H18O	1158	B. multifida	[47]
23	pinocarvone	C10H14O	1164	B. multifida	[45]
24	borneol	C10H18O	1168	B. multifida	[47]
25	terpinen-4-ol	C10H18O	1177	B. multifida	[45]
26	α-terpineol	C10H18O	1189	B. multifida	[43,45,47]
				B. orphanidis
27	myrtenal	C10H14O	1197	B. multifida	[45]
28	decanal	C10H20O	1204	B. multifida	[47]
29	trans-carveol	C10H16O	1217	B. multifida	[45]
30	carvone	C10H14O	1242	B. multifida	[45,47,48]
				B. heterostemon
31	geraniol	C10H18O	1255	B. heterostemon	[48]
32	linalyl acetate	C12H20O2	1257	B. orphanidis	[43]
33	isobornyl acetate	C12H20O2	1283	B. multifida	[47]
34	bornyl acetate	C12H20O2	1285	B. multifida	[45]
35	thymol	C10H14O	1290	B. multifida	[45]
36	(2E,4Z)-decadienal	C10H16O	1293	B. multifida	[47]
37	carvacrol	C10H14O	1302	B. multifida	[47]
38	(2E,4E)-decadienal	C10H16O	1316	B. multifida	[47]
39	δ-elemene	C15H24	1339	B. heterostemon	[48]
40	α-longipinene	C15H24	1352	B. heterostemon	[48]
41	eugenol	C10H12O2	1361	B. multifida	[47]
42	α-ylangene	C15H24	1374	B. multifida	[47]
43	geranyl acetate	C12H20O2	1381	B. heterostemon	[48]
44	β-elemene	C15H24	1391	B. multifida	[43,45,48]
				B. orphanidis
				B. heterostemon
45	tetradecane	C14H30	1400	B. multifida	[46]
46	isocaryophyllene	C15H24	1408	B. multifida	[47]
47	cis-caryophyllene	C15H24	1409	B. heterostemon	[48]
48	α-gurjunene	C15H24	1412	B. orphanidis	[43]
49	β-caryophyllene	C15H24	1416	B. multifida	[43,44,45,47,48]
				B. orphanidis
				B. heterostemon
50	α-bergamotene	C15H24	1418	B. heterostemon	[48]
51	β-duprezianene	C15H24	1424	B. multifida	[47]
52	γ-elemene	C15H24	1431	B. multifida	[45,48]
				B. heterostemon
53	α-humulene	C15H24	1449	B. multifida	[45,48]
				B. heterostemon
54	β-farnesene	C15H24	1457	B. multifida	[44,45,47,48]
				B. heterostemon
55	allo-aromadendrene	C15H24	1462	B. multifida	[44,48]
				B. heterostemon
56	α-amorphene	C15H24	1480	B. heterostemon	[48]
57	germacrene D	C15H24	1485	B. multifida	[45,48]
				B. heterostemon
58	(E)-β-ionone	C13H20O	1486	B. multifida	[47]
59	cis-β-guaiene	C15H24	1487	B. heterostemon	[48]
60	epi-cubebol	C15H26O	1495	B. multifida	[47]
61	bicyclogermacrene	C15H24	1495	B. multifida	[45]
62	α-selinene	C15H24	1498	B. heterostemon	[48]
63	germacrene A	C15H24	1501	B. heterostemon	[48]
64	β-bisabolene	C15H24	1505	B. multifida	[47]
65	α-farnesene	C15H24	1507	B. multifida	[44]
66	γ-cadinene	C15H24	1512	B. multifida	[44,47]
67	δ-cadinene	C15H24	1522	B. multifida	[44,45,47]
68	d-cadinene	C15H24	1525	B. heterostemon	[48]
69	guaia-3,9-diene	C15H24	1534	B. heterostemon	[48]
70	α-cadinene	C15H24	1536	B. multifida	[44,48]
				B. heterostemon
71	nerolidol	C15H26O	1538	B. multifida	[45,48]
				B. heterostemon
72	eudesma-3,7(11)-diene	C15H24	1545	B. heterostemon	[48]
73	elemol	C15H26O	1552	B. multifida	[44]
74	elixene	C15H24	1559	B. heterostemon	[48]
75	germacrane B	C15H24	1563	B. orphanidis	[43]
76	(E)-nerolidol	C15H26O	1565	B. multifida	[44,45,46,47]
77	spathulenol	C15H24O	1578	B. multifida	[43,45,47]
				B. orphanidis
78	caryophyllene oxide	C15H24O	1583	B. multifida	[43,44,45,47,48]
				B. orphanidis
				B. heterostemon
79	viridiflorol	C15H26O	1594	B. multifida	[44]
80	hexadecane	C16H34	1600	B. multifida	[46]
81	guaiol	C15H26O	1601	B. multifida	[47]
82	humulene epoxide II	C15H24O	1610	B. multifida	[44]
83	β-elemenone	C15H22O	1612	B. heterostemon	[48]
84	dillapiole	C12H14O4	1624	B. multifida	[47]
85	τ-cadinol	C15H26O	1635	B. multifida	[44,45]
86	epi-α-cadinol	C15H26O	1642	B. multifida	[43,47]
				B. orphanidis
87	α-eudesmol	C15H26O	1656	B. multifida	[44,47]
88	α-bisabolol oxide B	C15H26O2	1658	B. orphanidis	[43]
89	bulnesol	C15H26O	1671	B. multifida	[44,47]
90	(Z)-α-santalol	C15H24O	1674	B. orphanidis	[43]
91	cis-β-elemenone	C15H22O	1678	B. heterostemon	[48]
92	α-bisabolol	C15H26O	1688	B. multifida	[43,44,47,48]
				B. orphanidis
				B. heterostemon
92	germacrone	C15H22O	1699	B. heterostemon	[48]
94	(E)-nerolidol acetate	C15H26O	1714	B. multifida	[44,47]
95	(2E,6E)-farnesol	C15H26O	1727	B. multifida	[44,47]
96	octadecane	C18H38	1800	B. multifida	[46,47]
97	neophytadiene	C20H38	1836	B. multifida	[46]
98	6,10,14-trimethyl-2-pentadecanone	C18H36O	1845	B. multifida	[44,46,47]
99	nonadecane	C19H40	1900	B. multifida	[47]
100	farnesyl acetone	C18H30O	1917	B. multifida	[44,47]
101	methyl linolenate	C19H32O2	2098	B. multifida	[47]
102	phytol	C20H40O	2124	B. multifida	[44,47]
104	mandenol	C20H36O2	2148	B. multifida	[44]
104	ethyl linolenate	C20H34O2	2162	B. multifida	[44,47]
105	10-cyclohexyl-nonadecane	C25H52	2312	B. multifida	[44]
106	pentacosane	C25H52	2517	B. multifida	[44]
107	n-heptacosane	C27H56	2682	B. multifida	[44]
108	octacosane	C28H58	2791	B. multifida	[44]
109	nonacosane	C29H60	2894	B. multifida	[44]
110	vitamin E	C29H50O2	3138	B. multifida	[44]
111	n-docosane	C22H46	—	B. multifida	[44]
112	epizonaren	C15H24	—	B. multifida	[44]

In comparison with B. multifida, the investigations of chemical compositions of essential oils extracted from B. heterostemon and B. orphanidis were still limited. Forty compounds (82.43% of total essential oil) were identified in the essential oil of aerial parts of B. heterostemon, mainly containing β-caryophyllene (33.79%), elixene (5.09%), β-elemenone (4.45%), germacrene D (3.64%), camphor (3.34%), α-bisabolol (3.30%) and geraniol (3.27%) [48]. Thirteen components constituting 98.12% of the essential oil extracted from the aerial parts of B. orphanidis were detected, and the major chemical constituents included cis-limonene oxide (47.90%), β-caryophyllene (9.70%) and α-bisabolol (8.23%) [43]. Furthermore, oxygenated monoterpenes (51.25%) were found to be predominated over other chemical types in the constituent composition of the B. orphanidis essential oil in this study [43].

5. 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.

6. Pharmacological Activities

6.1. In Vivo Pharmacological Activities

6.1.1. 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.

6.1.2. Anti-Hypertensive and Hypoglycemic Effects

The compound N-3-methyl-2-butenyl urea (40) isolated from B. heterostemon displayed both analgesic and antihypertensive activities [11]. Numerous alkaloids from natural resources exhibited hypoglycemic effects [14]. B. heterostemon alkaloids showed significant hypoglycemic efficacy in streptozotocin-induced diabetic mice, with the optimum therapeutic dose at 5 mg/kg [59]. On the other hand, neither antihypertensive nor hypoglycemic activity was detected in any other Biebersteinia species. In addition, galegine (30), an isopentenyl guanidine, which was originally isolated from Galega officinalis and has significant hypoglycemic activity [60], was also detected in B. heterostemon [25]. In fact, the hypoglycemic drug metformin is a derivative of galegine [61,62,63,64].

6.1.3. 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.

6.1.4. 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.

6.2. In Vitro Pharmacological Activities

6.2.1. 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].

6.2.2. 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 IC50 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.

6.2.3. 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].

7. 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.