Structural and bioactive studies of terpenes and cyclopeptides from the Genus Rubia

Genus Rubia fell into about 70 species distributed widely around the world, a total of 36 species and 2 varieties were reported from China. The extracts and phytochemicals of Rubia plants had drawn considerable attention due to their potent bioactivities. As the two major ingredients from these plants, pentacyclic triterpenes and cyclopeptides were becoming a hot topic over the past twenty years for their remarkable anticancer, antioxidant and other effects. This paper compiled all 65 terpenes and 44 cyclopeptides with their distributions, physiological activities and melting points (or optical rotations) as reported in 85 references; besides, structure-activity relationships of these derivatives were briefly discussed. The information involved in this paper was expected to be meaningful for the further studies of the Genus Rubia.


Introduction
Genus Rubia that belongs to the Rubiaceae is one kind of perennial herbs; it falls into about 70 species distributed widely around the world, such as Western Europe, Northern Europe, Mediterranean Coast, Temperate Asia, Africa, Himalaya, as well as the regions from Mexico to Tropical America. A total of 36 species and 2 varieties have been reported from China [1]. Rubia species being one of the earliest plant resources possessed important commercial and medicinal values. Commercially, they were used as natural dye-stuffs in old days and improved commodity circulation; medically, these species being used as drugs were first recorded in the world famous pharmacy book of China, Divine Famer's Materia Medica, which has over 2000 years history [2]. According to many medical books, the roots of Rubia plants being reputed for their satisfactory efficacy were wildly used for the treatment of cancers, tuberculosis, rheumatism, hematemesis, metrorrhagia, epistaxis, contusion and menoxenia in the Chinese traditional medicine [3,4]. Besides, Indian folk medicine also comprised numerous prescriptions involved in Genus Rubia for healing wounds, inflammation, skin infections, and so on [5].
Within the last few decades, the extracts and phytochemicals of Rubia plants have drawn much attention due to their potent bioactivities. Studies on these plants led to the isolation of a series of bioactive ingredients including anthraquinones, naphthoquinones, terpenes, cyclopeptides and other constituents. Nine years ago, R. Singh etc. summarized a total of 33 terpenes and 19 cyclopeptides identified from 7 Rubia species [6]. Since then, researches on phytochemicals of Genus Rubia have achieved a lot and the lack of a comprehensive and lately review on this subject prompted us to gather much more new information. The previous review of us has introduced 148 anthraquinones and naphthoquinones together with their structure-activity relationships; herein we continue a general presentation of Rubia terpenes and cyclopeptides that exhibited remarkable anticancer, antioxidant and other effects. The present paper is dedicated to summarizing and updating a total of 65 terpenes and 44 cyclopeptides whose structures, distributions and properties are listed in the following tables and figures; furthermore, structure-activity relationships of these derivatives are briefly discussed.

Terpenes and their glycosides isolated from the Genus Rubia
Terpenes were not generally considered as major effective ingredients existed in medicinal Rubia plants, but they were also the earliest phytochemicals isolated from Genus Rubia. To our knowledge, people have got 65 terpene derivatives including 5 monoterpenes and 60 pentacyclic triterpenes. Compared with the similar work in 2004, this paper supplemented 32 new terpenes; plant sources of several compounds were also extended. What's more, we modified some previous information; for instance, β-sitosterol, daucosterol and stigmasterol should be classified under the steroids rather than the terpenes; three pentacyclic triterpenes named 'Rubiprasins A-C' were mistaken as 'Rubiprassins A-C' and Rubiprasin C's structure was also wrongly described. All of the 65 terpenes and their glycosidic derivatives isolated from 8 Rubia species are listed in Table 1 together with melting points (or optical rotations); the structures of these compounds are described in Additional file 1: Figure S1.
Except for compiling and summarizing the latest information, in the process of literature searching, we deem that some previous information is not credible. For instance, melting points of Rubiarbonone B (26) which roots in two different references should not be deviated too much [14,16,18]; it's also doubted that the structure of Rubiarboside F (34) was describe as IV's (49) [15,21]. More detailed information of Rubia triterpenes is listed in the following

Cyclopeptides isolated from the Genus Rubia
Studies on plant cyclopeptides have drawn considerable attention for their distinctive bicyclic structural features and significant antitumor activities. On the basis of the chemical skeletons and distributions in plants, N. H. Tan etc. proposed the systematic structural classification of plant cyclopeptides which were divided into two classes, five subclasses, and eight types [38]. Followed by the rule, Rubia cyclopeptides should be classified under the Rubiaceae-type cyclopeptides formed with one α-Dalanine, one α-L-alanine, three N-methyl-α-L-tyrosines and one other proteinogenic α-L-amino acid [39]. So far, people have got 44 cyclopeptide derivatives from the Genus Rubia. Compared with terpene derivatives, Rubia cyclopeptides have narrower sources and only existed in three species containing R. cordifolia, R. yunnanensis and R. akane. In general, according to these cyclopeptides' names, we may deduce their distributions; for instance, the cyclopeptides named Rubiyunnanins and RYs were reported from R. yunnanensis while the structures whose names were designated as RAs or others mainly came from R. cordifolia.

Biological activities of Rubia terpenes and Rubia cyclopeptides
Various crude fractions and purified compounds from the Genus Rubia exhibited a relatively wide range of biological activities [64][65][66][67][68][69]. Numerous experimental data verified that pentacyclic triterpenes possessed potent advantage on the aspects of antitumor, antiinflammatory, protecting liver, anti-HIV effects [70], while the most significant advantage of plant cyclopeptides was their underlying mechanisms of antitumor effects. As the two major ingredients existed in those Rubia plants, pentacyclic triterpenes and cyclopeptides were becoming a hot topic over the past twenty years for their remarkable effects. The following contents aims to offer the detailed statements of pharmacological activities of Rubia terpenes and cyclopeptides, especially their anticancer and antioxidant effects. Finally, all the bioactive terpenes and cyclopeptides together with the sketchy mechanisms are collected in Table 3.
Different from pentacyclic triterpenes' wide bioactivities, the effects of Rubia cyclopeptides mainly focused on inhibitory against tumors; and it's available to identify bioactive cyclopeptides. An efficient isolation method for antitumor cyclopeptides from Genus Rubia had been established by following the activity against murine tumor P388 leukemia that was successfully used to purify RAs I-IV (66-70) [40]. In a similar way, H. Itokawa etc. [41] found that the methanolic extract prepared from roots of R. cordifolia had a significant antitumor activity against Sarcoma 180 ascites and P388 leukemia in mice, from which RAs-III, -IV, -V (71) and -VII (74) were obtained. Recent years, monoclonal antibodies against RA-VII (74) had been also generated for screening of antitumor cyclopeptides. T. Hasuda etc. proved that they were useful for checking of the RA series compounds in the roots of R. cordifolia and R. akane [71].
Various data exhibited that different structural residues of Rubia cyclopeptides seemed to be closely related with their anticancer effects. Studies on the effect of RA-XVII (84) on cytotoxicity and conformation showed that although the structure exhibited little effect on the conformation of the molecule, it might decrease the activity as the side chain of residue 1 grew longer [47]. On the basis of several cyclopeptide derivatives, the typical type II β-turn structure and the aromatic side chain of Tyr-3 over this turn were also considered to play a very important role in its antitumor activities [42]. RA-X (77) containing a glutamic acid at residue 2 possessed strong antitumour effect on P388 while RA-IX (76) with conformation restricted to a type II β-turn at residues 2 and 3 showed almost no antitumour activities [43]. RAs XI-XIV (78-81) had potent antitumor activity against P388, especially the compound 79 whose residue 2 was methyl. The conformation of cis N-methyl amide bond between residues 2 and 3 also played an important role in the antitumour activities of RAs [50]. RA-XVIII (85) was another natural peptide of the RA-series in which the benzene ring of Tyr-6 was hydroxylated and its cytotoxicity against P-388 was 0.012 μg/ml [51]. RA-XIX, -XX, -XXI and -XXII (86-89) had been evaluated for their cytotoxicity against P388, with RA-VII (74) as reference; for the compounds having a methoxyl group in Tyr-6, the order of cytotoxicity was 74 > 87 > 86. Compound 89 possessing a hydroxyl group in its Thr-2 was less cytotoxic than 88. It seemed that the cytotoxicity decreased with increased in the length of the carbon side chain or introduction of a polar functionality at this R. cordifolia [59] location [52]. RA-XXIII (90) and RA-XXIV (91) exhibited just moderate cytotoxicity against P-388 with IC 50 values of 0.16 and 0.48 mg/ml, respectively [53]. T. Koizumi etc. had ever applied RA-VII (74) for the treatment of tumors in mice; daily intraperitoneal injection of RA-VII (1.5 or 3 mg/kg/day) had no toxic effects on those animals, but significantly and dose dependently inhibited the growth of Lewis lung carcinoma cells previously inoculated into the mice [72]. Compared with the various sources and remarkable effects of RAs, cyclopeptides named after Rubiyunnanin only existed in R. yunnanensis and their efficacies on tumors were relatively weaker. Cytotoxicities of Rubiyunnanin A (92) and B (93) against the 11 cancer cell lines were measured by SRB assay, but only 93 possessed moderate cytotoxicities [54]. Rubiyunnanins C-H (94-99) and RA-V, RA-I, RA-XXIV, RA-XII, RY-II not only exhibited cytotoxicities against the same cell lines with IC 50 values ranging from 0.001 to 56.24 μM, but also exerted inhibitions against NO production in LPS and IFN-c-induced RAW 264.7 murine macrophages with IC 50 values ranging from 0.05 to 12.68 μM [55]. RYs I-III (100-102), RA-700 (104), and RC-18 (105) possessed only medium activities against P388 [56][57][58]60,61]. The newly identified compounds 106-108, and RA-V, VII were evaluated for their cytotoxic activities against HL-60 and HCT-116 while only the latter had potent cytotoxicities [62]. Moreover, compound 109 exhibited cytotoxic effects on HL-60 cells, but which was much weaker than those of RA-XXIV and RA-VII [63].

Inducing cell apoptosis
Researchers paid much attention on anticancer mechanisms of maslinic acid (9), ursolic acid (11) and oleanolic acid (12) over the past decade years. No obvious correlation could be observed between cytotoxicity and inhibitory activity of DNA relaxation and decatenation by DNA topoisomerases I and II [24]. Oleanolic acid had selective inhibitory activity against DNA topoisomerase II compared with DNA topoisomerase I but weak cytotoxicity against HT-29, MCF-7 and HepG2 [26]. Maslinic acid was able to induce caspase-dependent apoptosis in human coloncancer cells via the intrinsic mitochondrial pathway [73,74]; it could also potentiate anti-tumor activities of TNF-α and inhibit pancreatic tumor growth and invasion by activating caspase-dependent apoptotic pathway and by suppressing NF-κB activation and its downstream gene expression [75]. Inhibition of Protein kinase C (PKC) that was related to the tumor development might lead to inhibition of cells growth and spreading of cancer cells, while maslinic acid acted as a PKC inhibitor. Ursolic acid potentiated TRAIL-induced apoptosis through activation of reactive oxygen species and  Anti-HIV 9, 11 Notice: Compounds' Code Names are involved in these derivatives listed in Table 1 and Table 2.
JNK-mediated up-regulation of death receptors and downregulation of decoy receptor 2 and cell survival proteins [76,77]; it was also considered as a novel blocker of STAT3 activation that might have a potential in prevention and treatment of multiple myeloma and other cancers [78]. Ursolic acid and oleanolic acid possessed markedly apoptotic effects on four cell lines via increasing DNA fragmentation, decreasing mitochondrial membrane potential, lowering Na + -K + -ATPase activity and elevating caspase-3 and caspase-8 activities; they could suppress cell adhesion and reduce the production of VEGF and ICAM-1 in these cell lines [79].

Antiangiogenic effect
Compared with the inhibition of Rubia cyclopeptides on cell lines, antiangiogenic effect of them were rarely involved. Sato and his coworkers demonstrated the antiangiogenic activity of RA-VII (74) on the proliferation, migration, stress fiber formation of BAEC and effects on mouse corneal angiogenesis [72]. Last year, G. G. L. Yue etc. discovered RA-V (71) also possessed the activity in HUVEC and HMEC-1 with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway; however, RA-V might regulate different signalling pathways in various endothelial cells [80].

Antioxidant effects of Rubia terpenes and Rubia cyclopeptides
Free radical NO was reported to be implicated in lots of physiological and pathological processes including vasodilation, nonspecific host defense and chronic or acute inflammation. It was produced by the oxidation of L-arginine under NO synthase (NOS). In the family of NOS, iNOS was particularly involved in pathological aspects with overproduction of NO, which could be expressed in response to pro-inflammatory agents such as interleukin-1β, tumor necrosis factor-a and lipopolysaccharide (LPS) in various cell types such as macrophages, endothelial cells and smooth muscle cells [21]. The bioactivities of rubianols on NO production from LPS-activated macrophages had been examined. Rubianol-d (43) and rubianol-e (44) exhibited the inhibition without cytotoxicity in the MTT assay whose efficacy was equivalent to L-NMMA's; rubianol-c (42), rubiarbonols A (16) and F (21), rubiarbonone C (27) showed both cytotoxic and inhibitory activities on NO production [19]. RA-V (71) and RA-XII (79) were demonstrated to possess potent effects on iNOS induction, and the suppression was closely related to their inhibitions of NO [21]. Inhibitory effects of Rubiyunnanin A-H (92-99) and RA-V, RA-I, RA-XXIV, RA-XII, RY-I were also evaluated; all of the compounds except 92 exhibited activity against NO production in LPS [54].
Other effects of Rubia triterpenes Antiplatelet aggregation effect Among compounds 16,17 and 25,Rubiarbonol B (17) exhibited the most potent inhibition against arachidonic acid-induced and collagen-induced platelet aggregation at 150 μM, while Rubiarbonol A (16) and Rubiarbonone A (25) promoted platelet aggregation at the high doses and possessed antiplatelet aggregation activity at the lower concentrations. This result might be consistent with a basic tenet of Traditional Chinese Medicine, in that variations of the dose of prescriptions induced either stimulatory or inhibitory effects [15].

Antimicrobial effects
Antibacterial activity against Staphylococcus aureus and antifungal activity against Candida albicans of triterpenes from R. yunnanensis were evaluated using the turbidimetric method; compound 22 exhibited inhibition on both of the two strains [22]. Besides, ursolic acid (11) and oleanolic acid (12) possessed fairly high antimicrobial activities but were weaker than clinical antimicrobial drugs'; however, both of them exhibited low toxicity and might be used for treatment of infections by Vancomycin-Resistant Enterococci [81].

Antiobesity effect
Excess visceral adiposity might predispose to chronic diseases like hypertension and type-II diabetes with a high risk for coronary artery disease. Adipose tissue secreted cytokines and oxidative stress played an important role in the chronic disease progression. Triterpene derivatives had abilities to regulate glucose and lipid metabolism. C. L. Melo etc. [31] verified that oleanolic acid (12) could ameliorate visceral adiposity and improve glucose tolerance in mice and had an antiobesity potential through modulation of carbohydrate and fat metabolism. Much attention was also focused on food that might be beneficial in preventing diet-induced body fat accumulation and possibly reducing the risk of diabetes and heart disease; then ursolic acid (11) was demonstrated to have potent bioactivities improving certain metabolic parameters associated with diets high in saturated fats and obesity [82].

Antidiabetic effect
As a natural and low toxic compound, maslinic acid (9) elicited excellent outcomes without inducing the side effects. It exhibited significant glucose-lowering and hypoinsulinemic effects in KK-A y mice and might hold great promise as a natural therapeutic agent for treatment of type-II diabetes [83]. Furthermore, both ursolic acid (11) and oleanolic acid (12) markedly suppressed renal aldose reductase activity and enhanced the activity of glyoxalase I, which contributed to decrease renal AGEs formation and improve renal functions; therefore, supplement of oleanolic acid and ursolic acid or foods rich in these compounds might be helpful for the prevention or treatment of diabetic kidney diseases [84].

Conclusions
To our knowledge, the reported Rubia terpenes distributed in 8 species and most of them derived from R. yunnanensis. The triterpenes were designated under a series of names including rubiarbonol, rubiarbonone, rubiarboside, rubiprasin and rubianol; among them, oleanane-type and fernane-type triterpenes accounted for overwhelming majority. Rubia cyclopeptides had narrower sources and existed in only 3 species including R. cordifolia, R. yunnanensis and R. akane. These terpenes and cyclopeptides, especially the latter, possessed excellent anticancer effects; maslinic acid, ursolic acid, oleanolic acid and several cyclopeptides containing RA-V and RA-VII drew relatively more attention than any other compound. From Table 3 and Additional file 1: Figure S1, it may be inferred that the Rubia triterpenes owning a free hydroxyl group on third carbon atom exhibited relatively strong inhibitory on cell lines; besides, fernane-type triterpenes possessed promising antioxidant effects. As for Rubia cyclopeptides, increase in the length of carbon side chain at residues 1, 2 and introduction of polar groups at residue 2 might decrease structure's cytotoxicity; N-methyl amide bond between residues 2 and 3, together with aromatic side chain of residue 3 over β-turn played pivotal role in the antitumor activities. Beyond that, the orientation of one or both of the Tyr-5 and Tyr-6 phenyl rings was also essential to express activities, while structures possessing two rings formed between Tyr-5 and residue 6 via a phenolic oxygen linkage and a new carbon bond only possessed moderate cytotoxicities.
Up to now, phytochemical investigations of the Genus Rubia have resulted in identification of various chemical components including anthraquinones, naphthoquinones, terpenes and cyclopeptides. This paper dedicated to compiling all 65 terpenes derivatives and 44 cyclopeptides derivatives from 8 Rubia species. Together with the previous work, we have already finished summarizing a total of 257 compounds from 12 Rubia species with their bioactivities. However, taking into account the fact that 70 species belong to the Genus Rubia spreading around the world, only 17% of them have been phytochemically investigated, the Genus Rubia still remains to be a potential resource to research. Furthermore, as one kind of natural dyestuffs, resource exploration of Genus Rubia is also an asset to economic development and waits for its full exploitation.