A Review of the Botany, Phytochemistry, Pharmacology and Toxicology of Rubiae Radix et Rhizoma.

Rubia cordifolia Linn (Rubiaceae) is a climbing perennial herbal plant, which is widely distributed in China and India. Its root and rhizome, Rubiae Radix et Rhizoma (called Qiancao in China and Indian madder in India), is a well known phytomedicine used for hematemesis, epistaxis, flooding, spotting, traumatic bleeding, amenorrhea caused by obstruction, joint impediment pain, swelling and pain caused by injuries from falls. In addition, it is a kind of pigment utilized as a food additive and a dye for wool or fiber. This review mainly concentrates on studies of the botany, phytochemistry, pharmacology and toxicology of this Traditional Chinese Medicine. The phytochemical evidences indicated that over a hundred chemical components have been found and isolated from the medicine, such as anthraquinones, naphthoquinones, triterpenoids, cyclic hexapeptides and others. These components are considered responsible for the various bioactivities of the herbal drug, including anti-oxidation, anti-inflammation, immunomodulation, antitumor, effects on coagulation-fibrinolysis system, neuroprotection and other effects. Additionally, based on these existing results, we also propose some interesting future research directions. Consequently, this review should help us to more comprehensively understand and to more fully utilize the herbal medicine Rubiae Radix et Rhizoma.

1. Introduction

Rubia cordifolia Linn (R. cordifolia, Rubiaceae) is a climbing perennial herbal plant widely distributed in China and India. As a medicinal plant, it was firstly recorded in a formula with endoconch of Sepiella maindroni in “Huangdi Neijing”, the most important ancient book in Chinese Traditional Medicine (TCM). Rubiae Radix et Rhizoma (RRR), the dried root and rhizome of R. cordifolia, is a famous TCM used for thousands of years. The medicinal part of R. cordifolia was specificly presented for the first time in “Shennong Bencaojing”, another classic work on plants and their uses. Since then, this phytomedicine has been employed for hematemesis, epistaxis, flooding, spotting, traumatic bleeding, amenorrhea caused by obstruction, joint impediment pain, swelling and pain caused by injuries from falls [1]. With its excellent activity to cool blood, eliminate stasis, stop bleeding and unblock meridians, it has been considered in “Bencao Gangmu” and other ancient medical books as an important herbal drug for curing the syndromes caused by blood heat. Thus, RRR is still listed in the Chinese Pharmacopeia [1]. During the past decades, more than one hundred chemical compounds such as anthraquinones, naphthoquinones, cyclic hexapeptides, terpenoids, polysaccharides and other compounds have been found in RRR, isolated and identified [2,3,4,5,6,7,8,9,10]. The range of its pharmacological activity also has been widened, covering antioxidation, neuroprotection, anti-inflammation, antitumor, and immunomodulation effects, etc [3,11,12,13,14,15,16,17].

Additionally, R. cordifolia is a well-known source of red plant dye for clothes and food items with a long history of use in China and India [18,19,20,21,22]. The earliest record of this was in “Shijing”, an ancient book about over 2500 years ago. In China, RRR has been listed by the Ministry of Health as one of the substances that can be used in dietary supplements.

With the developments of science and technology and the introduction of many advanced experimental methods and instruments during the recent decades, a large number of studies have been performed on many aspects of RRR and a lot of achievements have been made, which were reported in many literatures. So, to summarize these studies and findings, we aim with this paper to provide a comprehensive and up-to-date review of Rubiae Radix et Rhizoma that covers the studies of its botany, phytochemistry, pharmacology and toxicology.

2. Botany

According to the Chinese Pharmacopeia [1], R. cordifolia Linn is the sole and authentic plant source of Rubiae Radix et Rhizoma. At present, R. cordifolia L. varstenophylla Franch, R. cordifolia L. varherbacea Chun et How, R. cordifolia L. varpratensis Maxim and R. cordifolia L. varmollis Chun et How are considered as the indigenous varieties of R. cordifolia Linn [23]. In China, it is widely distributed in most regions, including the provinces of Shaanxi, Henan, Anhui, Hebei, Shandong, Hubei, Jiangsu, Zhejiang, and so on. Among them, Weinan Shannxi and Songxian Henan are the most fundamental production areas, famous for their high production volume and quality [24]. In some areas of China, R. schumanniana pritz., R. yunnanensis Diels, R. membranacea Diels, R. tinctorum L. and so on are used as folk medicines rather than R. cordifolia Linn. Nevertheless, neither of them is the authentic one [25].

R. cordifolia is a climbing perennial herbal plant. The roots, which cluster in the soil, are aubergine or orange-red. The elongating and rough stems slightly lignify at the base. The branches are four-edge shaped and there are some anatropousspinules on the edges. The papery leaves are ovate or ovate-lanceolate and 2–6 cm long, 1–3 cm wide [24]. Sometimes there are sparse bristles on the rough-surfaced leaves. The cymes are in the axils or on the tops and are in the shapes of large and loose cones. The subsphaeroidal fruits with smooth surfaces, about 5 cm in diameter, are black or black-purple when ripening. Growing at the altitude of 570–1800 m, R. cordifolia is found along the roadside or riverside, on hillsides and in valleys [23]. Generally, the root and rhizome are collected in spring or autumn in the third or fourth year after cultivation [25]. Figure 1 shows R. cordifolia and Rubiae Radix et Rhizoma.

Figure 1

Rubia cordifolia Linn (A) and Rubiae Radix et Rhizoma (B).

3. Phytochemistry

More than a hundred components, which belong to anthraquinones, naphthoquinones, cyclic hexapeptides, terpenoids and other classes of compounds, have been found and isolated from RRR. Some of them have been proved responsible for the pharmacological activities in many studies. The compounds reported in the literature are listed in Table 1.

Table 1

Compounds in Rubiae Radix et Rhizoma.

Category	No.	Compound	Molecular Formula	Reference
Anthraquinones	1	Alizarin	C14H8O4	[6,9,26,27,28]
	2	Alizarin 2-methyl ether	C15H10O4	[29]
	3	Lucidinprimeveroside	C26H28O14	[6,9,30]
	4	Munjistin	C15H8O6	[7,31]
	5	Nordamnacanthal	C15H8O5	[32]
	6	Pupurin	C14H8O5	[9,28,31]
	7	Physcion	C16H12O5	[32,33]
	8	Ruberythric acid	C25H26O13	[6,9,28,30]
	9	Rubiacordone A	C23H22O10	[34]
	10	Rubiadin	C15H10O4	[7,9,35]
	11	Soranjidiol	C15H10O4	[29]
	12	Tectoquinone	C15H10O2	[9,36,37,38]
	13	Xanthopurpurin	C14H8O4	[7,9,28,36]
	14	1-Hydroxyanthraquinone	C14H8O3	[30]
	15	1-Hydroxy-2-methylanthraquinone	C15H10O3	[6,7,9,32,36,37]
	16	1,4-Dihydroxy-6-methylanthraquinone	C15H10O4	[32]
	17	1-Hydroxy-2-methoxyanthraquinone	C15H10O4	[35]
	18	1,3-Dimethoxy-2-carboxylanthraquinone	C17H12O6	[35]
	19	1,3,6-Trihydroxy-2-methylanthraquinone	C15H10O5	[6,7,8,26,30,39]
	20	1,4-dihydroxy-2-methylanthraquinone	C15H10O4	[9,36,40]
	21	1,4-Dihydroxy-2,3-dimethylanthraquinone	C16H12O4	[41]
	22	1,5-Dihydroxy-2-methylanthraquinone	C15H10O4	[40]
	23	1,3-Dihydroxy-2-ethoxymethylanthraquinone	C17H14O5	[6,41]
	24	1,4-Dihydroxy-2-methy-5-methoxylanthraquinone	C16H12O5	[9]
	25	1-Acetoxy-3-methoxyanthraquinone	C17H12O5	[29]
	26	1-Hydroxy-3-carbomethoxyanthraquinone	C16H10O5	[36]
	27	1-Hydroxy-2-hydroxymethylanthraquinone	C15H10O4	[8,36]
	28	1-Hydroxy-3-hydroxymethylanthraquinone	C15H10O4	[41]
	29	1-Hydroxy-3-ethylanthraquinone	C16H12O3	[41,42]
	30	1-Hydroxy-2,7-dimethylanthraquinone	C16H12O3	[2]
	31	1,2,4,6-Tetrahydroxyanthraquinone	C14H8O6	[43]
	32	1,2,4-Trihydroxylanthraquinone	C14H8O5	[30]
	33	2-Hydroxy-6-methylanthraquinone	C15H10O3	[2]
	34	2,6-Dihydroxyanthraquinone	C14H8O4	[2]
	35	1,3,6-Trihydroxy-2-methyl-9,10-anthraquinone-3-O-β-glucoside	C21H20O10	[7,30]
	36	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-α-rhamnosyl-(1→2)-β-glucoside	C27H30O14	[6,7,39]
	37	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-(3′-O-acetyl)-α-rhamnosyl-(1→2)-β-glucoside	C29H32O15	[7]
	38	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-(6′-O-acetyl)-α-rhamnosyl-(1→2)-β-glucoside	C29H32O15	[6,7]
	39	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-(4′,6′-O-diacetyl)-α-rhamnosyl-(1→2)-β-glucoside	C31H34O16	[7]
	40	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-(3′,6′-O-diacetyl)-α-rhamnosyl-(1→2)-β-glucoside	C31H34O16	[7,39]
	41	1,3,6-Trihydroxy-2-methylanthraquinone-3-O-(6′-O-acetyl)-α-xylopyranosyl-(1→2)-β-glucoside	C28H30O15	[30]
	42	1,8-Dihydroxy-11,20(15-pentylnaphthaquinonyl) phenanthrene	C26H20O4	[42]
Naphthoquinones	43	Mollugin	C17H16O4	[6,7,9,36,44,45]
	44	Furomollugin	C14H10O4	[29,44,45,46]
	45	2′-Hydroxymollugin	C17H16O5	[8,38]
	46	2′-Methoxymollugin	C18H18O5	[8]
	47	1′,2′-Dihydroxydihydromollugin	C17H18O6	[8]
	48	1′-Methoxy-2′-hydroxydihydromollugin	C18H20O6	[8]
	49	Epoxymollugin	C17H16O5	[29]
	50	Dihydromollugin	C17H18O4	[7,33]
	51	3-Prenyl-5-methoxynaphthoquinone	C16H16O3	[40]
	52	3-Prenyl-8-methoxynaphthoquinone	C16H16O3	[40]
	53	2-Carbamoyl-3-hydroxynaphthoquinone	C11H7NO4	[36]
	54	2-Carbnmoyl-3-methoxynaphthoquinone	C12H9NO4	[36]
	55	Dehydro-α-lapachone	C15H12O3	[36]
	56	2-Carboxymethyl-3-prenyl-2,3-epoxynaphthoquinone	C17H16O5	[8,29,33,47]
	57	2-Carbomethoxy-3-(3′-hydroxy)isopentyl-1,4-naphthohydroquinone 4-O-β-glucoside	C23H30O10	[7]
	58	2-Carbomethoxy-3-prenyl-1,4-naphthohydroquinone 1,4-di-O-β-glucoside	C29H38O14	[7]
	59	5-Hydroxy-2-[7-hydroxy-4-(1-hydroxy-1-methylethyl)-2-methyl-6-oxo-2,3,3a,6-tetrahydro-4H-1,5-dioxabenzo-[de]anthracen-2-yl]-naphtho[1,2-b]furan-4-carboxylic acid methyl ester	C33H28O9	[8]
	60	6-Hydroxy-2-(5-hydroxy-4-methoxycarbonyl-naphtho-[1,2-b]furan-2-yl)-2-methyl-3,4-dihydro-2H-benzo[h]-chromene-5-carboxylic acid methyl ester	C30H24O8	[8]
Cyclic hexapeptides	61	RA-I	C40H48N6O10	[3,48]
	62	RA-II	C40H48N6O9	[48]
	63	RA-III	C41H50N6O10	[3,48]
	64	RA-IV	C41H50N6O10	[48]
	65	RA-V	C40H48N6O9	[3,48]
	66	RA-VI	C41H50N6O10	[49]
	67	RA-VII	C41H50N6O9	[3,48]
	68	RA-VIII	C41H50N6O10	[49]
	69	RA-IX	C43H51N6O10	[50]
	70	RA-X	C43H52N6O11	[50]
	71	RA-XI	C42H50N6O11	[51]
	72	RA-XII	C46H58N6O14	[51]
	73	RA-XIII	C48H60N6O16	[51]
	74	RA-XIV	C48H58N6O15	[51]
	75	RA-XV	C48H60N6O15	[52]
	76	RA-XVI	C48H60N6O16	[52]
	77	RA-XVII	C41H50N6O9	[53]
	78	RA-XVIII	C41H50N6O10	[54]
	79	RA-XIX	C44H57N6O9	[55]
	80	RA-XX	C42H52N6O9	[55]
	81	RA-XXI	C41H50N6O9	[55]
	82	RA-XXII	C41H50N6O10	[55]
	83	RA-XXIII	C43H53N7O10	[56]
	84	RA-XXIV	C42H51N7O10	[56]
	85	Rubicordin A	C46H60N6O14	[3]
	86	Rubicordin B	C47H62N6O14	[3]
	87	Rubicordin C	C42H54N6O9	[3]
	88	Rubiyunnanin B	C46H58N6O14	[3]
	89	RY-II	C47H60N6O14	[3]
	90	neo-RA-V	C40H48N6O9	[5]
	91	allo-RA-V	C40H48N6O9	[5]
	92	O-seco-RA-V	C40H50N6O9	[5]
	93	O-seco-RA-XXIV	C42H53N7O10	[4]
	94	RAI-III	C41H50N6O10	[57]
	95	RAI-VI	C41H50N6O10	[57]
	96	RA-dimer A	C80H94N12O18	[58]
Triterpenoids	97	Oleanolic acid	C30H48O3	[29,33]
	98	Oleanolic aldehyde acetate	C32H50O3	[46]
	99	Rubiarbonol A	C30H50O4	[43,59]
	100	Rubiarbonol B	C30H50O3	[43]
	101	Rubiatriol	C30H50O3	[26]
	102	Rubicoumaric acid	C39H54O6	[10]
	1037	Rubifolic acid	C30H48O4	[10]
	104	Rubiprasin A	C32H52O5	[60]
	105	Rubiprasin B	C32H52O4	[60]
	106	Rubiprasin C	C32H50O5	[60]
	107	Ursolic acid	C30H48O3	[41]
	108	3-β-Friedelinol	C30H52O6	[61]
Other compounds	109	β-Sitostenone	C29H48O	[43]
	110	β-Sitosterol	C29H50O	[33,41]
	111	5-Methoxygeniposidic acid	C17H24O11	[62]
	112	6-Methoxygeniposidic acid	C17H24O11	[46]
	113	3,5-di-(p-hydroxybenzyl)phenol	C20H18O3	[2]
	114	n-Heptadecane	C17H36	[2]
	115	n-Nonadecane	C19H40	[2]
	116	(+)-Lariciresinol	C20H24O6	[38]
Other compounds	117	3,3′-bis(3,4-Dihydro-4-hydroxy-6-methoxy-2H-1-benzopyran)	C20H22O6	[29]
	118	8-Hydroxy n-pentadecanyl decan-4-en-1-oate	C25H48O3	[2]
	119	n-Octacosanyl octa-1-oate	C36H72O2	[2]
	120	Rubilactone	C15H10O5	[41,45,63]
	121	Rubioncolin B	C31H24O10	[8]
	122	2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-5-ω-hydroxypropyl-7-methoxybenzofuran	C20H24O6	[43]
	123	Palmitic acid	C16H32O2	[41]
	124	Tricosanoic acid	C23H46O2	[41]
	125	Rubiasin A	C15H16O2	[38,64]
	126	Rubiasin B	C15H16O2	[64]
	127	Rubiasin C	C15H16O2	[64]
	128	Atraric acid	C10H12O4	[61]
	129	Vanillic acid	C8H8O4	[61]
	130	d-3-O-Methoxy-chiroinositol	C7H14O6	[61]
	131	Polysaccharide RPS-1	Not mentioned	[65]
	132	Polysaccharide RPS-2	Not mentioned	[65]
	133	Polysaccharide RPS-3	Not mentioned	[65]
	134	Polysaccharide QA2	Not mentioned	[66]

3.1. Anthraquinones

Anthraquinone is a well-known category of phytochemicals. Alizarin, munjistin, purpurin, rubiadin, tectoquinone and xanthopurpurin are the common ones. Among them, purpurin is one of the two chemical markers that serve to evaluate the quality of the herbal medicine in the Chinese Pharmacopeia [1]. In pharmacological studies, it has been proved to have anti-angiogenic [67], and anti-oxidant [68,69] functions. Besides, these anthraquinonesare often found combined with glucoside, xylosyl-(1→6)-glucoside (primeverose) or rhamnosyl-(1→2)-glucoside to form the corresponding anthraquinone glycosides. The structures are shown in Figure 2.

Figure 2

Structures of anthraquinones in Rubiae Radix et Rhizoma.

3.2. Naphthoquinones

Naphthoquinone is another category of phytochemicals in RRR. Mollugin is the representative on, reported to have anti-cancer [70,71], anti-inflammatory [17,72,73], and neuroprotective activities [72]. With a content of 10−3 g/g level or even 10−2 g/g level in RRR, it is designated as the other chemical marker by the Chinese Pharmacopeia [1]. Meanwhile, some derivatives of mollugin were isolated, too. The structures are shown in Figure 3.

Figure 3

Structures of naphthoquinones in Rubiae Radix et Rhizoma.

Besides the naphthoquinone monomers above, three naphthohydroquinone dimers were also isolated, which were chemically named as 6-hydroxy-2-(5-hydroxy-4-methoxycarbonylnaphtho-[1,2-b]furan-2-yl)-2-methyl-3,4-dihydro-2H-benzo[h]-chromene-5-carboxylic acid methyl ester, rubioncolin B and 5-hydroxy-2-[7-hydroxy-4-(1-hydroxy-1-methylethyl)-2-methyl-6-oxo-2,3,3a,6-tetrahydro-4H-1,5-dioxa-benzo-[de]anthracen-2-yl]-naphtho[1,2-b]furan-4-carboxylic acid methyl ester.

3.3. Cyclic Hexapeptides

Some Japanese researchers were the first to isolate four cyclic hexapeptides from R. cordifolia and R. akane while screening for anticancer compounds in phytomedicines. Up to now, twenty four cyclic hexapeptides have been found and isolated from RRR, which were named as RA I-XXIV. Each RA contains both 18-member ring and 14-member ring systems, which consist of some amino acids including N-methyl-O-methyl-l-tyrosine, pyroglutamic acid, l-alanine, d-alanine, etc. Among these RAs, RA-V and RA-VII were the dominating two, found at levels of nearly 100 μg/g, while the others represent less than 1 μg/g in RRR. Furthermore, in recent years, some analogues or precursors of RAs were also isolated from RRR, such as neo-RA-V, allo-RA-V, rubicordin A-C, O-seco-RA-V, O-seco-RA-XXIV, and so on. The structures are shown in Figure 4.

Figure 4

Structures of cyclic hexapeptides in Rubiae Radix et Rhizoma.

3.4. Triterpenoids

Among phytomedicines, oleanolic acid and ursolic acid are common triterpenoids. The two compounds also have been found and isolated in RRR. Rubiprasin A-C, rubiarbonol A and B, rubicoumaric acid and rubifolic acid and other triterpenoids were isolated, too.

3.5. Other Compounds

Other than anthraquinones, naphthoquinones, cyclic hexapeptides and triterpenoids, phytochemistry researchers have found and isolated many other chemical compounds from RRR, including some organic acids, polysaccharides, rubilactone, rubiasin A-C, β-sitostenone, β-sitosterol, 5-methoxygeniposidic acid, 6-methoxygeniposidic acid, and so on.

4. Pharmacology

In many early pharmacological studies, RRR has been proved to have various functions, such as radioprotective [74], antidiabetic [75], hepatoprotective [76], antitumor [48,50,51,52,54,77], antiplatelet [78] effects, and so on [44], which were reported in previous reviews [79,80]. However, in recent years, some new activities and mechanisms were found that had not been previously reviewed.

4.1. Effects on the Coagulation-Fibrinolysis System

In TCM, RRR is well known for its activity of cooling blood, eliminating stasis, stopping bleeding and unblocking meridians [1]. In our previous study, we produced a rat model with blood stasis using a subcutaneous injection of adrenaline plus an ice water bath. After the model rats were treated with RRR, whole blood viscosity and plasma viscosity decreased and content of fibrinogen increased. In addition, shortened prothrombin time, prolonged thrombin time and activated partial thromboplastin time in the model rats were rectified after treatment with RRR. Furthermore, compared to model rats, RRR treatment also downregulated thromboxane B2 (TXB2) levels and upregulated 6-keto-PGF1α levels [81]. Evidence of a study on the coagulation-fibrinolytic system of normal rats revealed that RRR decotion elevated the activity of tissue plasminogen activator (t-PA) [82].All these findings prove the effects of RRR on the blood system.

As we know, plasma hyaluronan-binding protein (PHBP) is a serine protease able to activate coagulation factor VII and prourokinase and to circulate as a single-chain form. With spermidine or heparin, it is autoproteolytically converted into an active two-chain form. In a screening investigation, purpurin was identified as a specific inhibitor of spermidine-induced autoactivation of PHBP [83].

4.2. Antitumor

RRR methanol extract (RRRME) was found to inhibit human laryngeal carcinoma cell (HEp-2 cell) proliferation and lactate dehydrogenase (LDH) release, to decrease reduced glutathione (GSH), glutathione S-transferase (GST) and protein levels, and to increase lipid peroxidation (LPO) in a dose-dependent manner. Further fluorescent microscopy and transmission electron microscopy confirmed this apoptotic effect [15].

Many studies have demonstrated that the cyclichexapeptides were the bioactive components responsible for the antitumor activity of RRR. The majority of the RA series compounds showed cytotoxicity against many cancer cells, including P-388 leukaemia cells [64], SGC-7901 human gastric adenocarcinoma cells, A-549 human non-small cell lung carcinoma cells, and Hela (human cervical carcinoma) cells [3].

Some small molecule compounds in RRR are also important antitumor compounds. As an Ames assay showed, alizarin was found effective in reducing his+ revertants induced by 4-nitro-o-phenylenediamine and 2-aminofluorene. Comet assay results indicated that DNA damage induced by H2O2 and 4-nitroquinoline-1-oxide could be reduced by anthraquinone too [84]. Mollugin was also proved to potentiate autophagic activity, induce growth inhibition and apoptosis of HN4 human oral cancer cells and SK-BR-3 breast cancer cells. It could upregulate the expression of mammalian target of rapamycin (mTOR), downregulate fatty acid synthase(FAS) gene expression and activate nuclear factor-E2-related factor 2 (Nrf2) with heme oxygenase-1 (HO-1)via some signaling pathways, such as phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and extracellular signal–regulated kinases (ERK). This naphthoquinone could also inhibit the activation of nuclear factor-κB (NF-κB) and NF-κB-dependent gene products involving antiapoptosis (Bcl-2 and Bcl-xl), invasion (MMP-9 and ICAM-1), and angiogenesis (FGF-2 and VEGF) [70,71,85]. 1-Hydroxy-2methylanthraquinone was another compound from RRR exhibiting cytotoxic effects on A375 malignant skin melanoma cells [86].

4.3. Immunomodulation

RRR ethanol extract (RRREE) was found to have protective effect against immunosuppression of Swiss albino mice induced by lead nitrate. This immunomodulation was considered associated to the increasing of macrophagocyte number and phagocyticindex, immunoglobulin levels and plaque-forming cell number [13]. In the serum of most peanut-allergy patients, there were increased levels of immunoglobulin E (IgE). After animals of a peanut-anaphylaxis mice model were treated with RRR aqueous extract, peanut-triggered anaphylactic reactions and plasma histamine levels decreased significantly, as did IgE production by a human B-cell line [14].

4.4. Anti-Inflammation

Pharmacological evidences showed that RRREE was able to ameliorate the lead nitrate-induced oxidative damage by improving the activities of superoxidedismutase (SOD) and catalase (CAT), increasing the content of GSH, and suppressing LPO [13]. In another study of indomethacin-induced enterocolitis in rats, some acute intestinal inflammation effects such as bowel wall thickening, mesenteric haemorrhage, mesentery adhesion and multiple mucosal ulcers of small intestine and colon emerged. Serum LDH activity increased, too. These disorders and changes were ameliorated after RRREE-treatment [87].

After RAW 264.7 macrophages were stimulated by LPS, the contents of NO, inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) and interleukin-6 (IL-6) increased significantly. However, mollugin-incubation brought some reduction of these inflammatory mediators [17]. In a screening investigation of some phytochemicals, physcion and 1-hydroxy-2methylanthraquinone showed amelioration on the damage to mouse peritoneal macrophages induced by lipo-polysaccharide (LPS) and interferon-γ (IFN-γ), which was mediated through inhibition of iNOS protein expression and reduction of NO content [86].

4.5. Neuroprotection

In a study of reserpine-induced orofacial dyskinesia, RRRME-treatment was able to significantly inhibit vacuous chewing movements, tongue protrusions, orofacial bursts, catalepsy and to increase locomotion and rearing in an open field test. Meanwhile, bioassay results revealed that RRRME-treatment increased the levels of SOD, CAT, GSH, inhibited LPO and elevated dopamine levels in the forebrain region, compared with the model. These findings demonstrated that the neuroprotection of RRRME was related to its antioxidant activity [88].

Mollugin was considered as a neuroprotective agent for glutamate-induced neurotoxicityin the mousehippocampal HT22 cell line. The neuroprotection may be mediated by the effects on suppression of pro-inflammatory mediators, up-regulation of the expression ofHO-1 and the activity of HO, nuclear accumulation of Nrf2 and activation of p38 mitogen-activated protein kinase (MAPK) pathway [72]. In the T-REx293human embryonic kidney cell line, Aβ(42)-EGFP (enhanced green fluorescent protein) plays a key role in Alzheimer’s disease. Abnormal accumulation of Aβ(42)-EGFP would lead to apoptosis of this cell line. With treatment by RRR polysaccharides, Aβ(42)-EGFP accumulation decreased and cell activity was restored dramatically. Moreover, RRR polysaccharides inhibited cytotoxicity of Aβ(42)-EGFP, which may be mediated by potentiated degradation of proteosome [11].

4.6. Antioxidation

Results of studies on the gastroprotective effect on aspirin plus pylorus-ligated ulcer indicated that RRRME and its chloroform fraction brought notable decreases of ulcer index, total acidity, protein, pepsin content of the gastric fluid and increase of the mucin content. Among the key antioxidant parameters, after treatment with RRRME or its chloroform fraction, were a significant reduction in LPO and elevations in CAT, SOD, and GSH. It was concluded that the protective mechanism could perhaps be partly attributed to the effects of the herbal medicine on oxidative stress [12].

In N-nitrosodiethylamine-induced hepatocellular carcinoma rats, the activities of serum marker enzymes including aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and LDH with the levels of LPO and hydroxyl radicals in liver increased significantly. The opposite trends were seen with the activities of the antioxidants including SOD, CAT, GSH, GST, glutathione peroxidase (GPx) in liver and the levels of mitochondrial enzymes like isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), α-ketoglutarate dehydrogenase (α-KGDH) and respiratory chain enzymes like nicotinamide adenine dinucleotide (NADH) dehydrogenase and cytochrome c oxidase. However, all these changes in the model group were markedly and dose-dependently ameliorated by treatment of RRRME, which indicated RRR perhaps could be used as an antioxidant for the treatment of some cancers [16]. Structure-radical scavenging activity relationship results demonstrated that hydroxyl groups on the benzene rings were essential to the radical scavenging function of hydroxylanthraquinone [89].

4.7. Other Pharmacological Functions

Besides the effects above, RRR have exhibited good effects in studies of anti-urolithiasis, anti-psoriasis, anti-nephrotoxicity, estrogenic and progestational activity, and so on [47,90,91,92,93,94,95] (Table 2).

Table 2

Other functions of Rubiae Radix et Rhizoma.

Function	Inducer	Test Drug	Model	Efficacy Evaluation	Reference
Anti-adipogenic activity		2-Carboxymethyl-3-prenyl-2,3-epoxynaphthoquinone	3T3-L1 preadipocytes	Induced MMP loss, caspase-3 activation	[54]
				Reduced differentiation-associated accumulation of intracellular lipid
				Downregulatedexpressions of CCAAT/enhancer binding protein-α, PPAR γ1, PPAR γ2, adiponectin
Anti-urolithiasis	Ethylene glycol	Ethanol extract	Male Wistar albino rats	Decreased calcium, oxalate levels and number of calcium oxalate crystals deposits in kidney tissue	[90]
Anti-psoriasis		Ethyl acetate fraction of ethanol extract	HaCaT cells	Decreased MMP	[91]
				Induced apoptosis
			Male BALB/c mice	Increased NGL, TGL and VET
Anti-nephrotoxicity	Cisplatin	Ethanol extract	Swiss albino mice	Decreased values of serum urea and creatinine	[92]
				Increased GPx, SOD and CAT
				Inhibited LPO in kidney and liver
Estrogenic and progestational activity		Ethyl acetate precipitate of methanol extract	Old female albino rats	Increased the regularity of the estrous cycle	[93]
				Increased uterine weight and foetal survival
Anti-osteoclastogenesis	NF-κB ligand	Mollugin	Mice BMMs	Inhibited osteoclast differentiation	[94]
				Reduced the phosphorylation ofMAP kinase, Akt, and GSK3β
				Inhibited expression of c-Fos, NFATc1,
				OSCAR, TRAP, DC-STAMP, OC-STAMP, integrin αν, integrin β3, cathepsin K, and ICAM-1
Anti-HIV	HIV-1NL4.3	Ethyl acetate extract	CEM-GFP cells	Reduced viral production	[95]

BMMs: bone marrow macrophages; DC-STAMP: dendritic cell-specific transmembrane protein; MMP: mitochondrial membrane potential; NFATc1: nuclear factor of activated T-cells cytoplasmic 1; NGL: number of granular layer; OC-STAMP: osteoclast stimulatory transmembrane protein; OSCAR: osteoclast-associated receptor; PPAR: peroxisome proliferator-activated receptor; TGL: vertical thickness of granular layer; TRAP: tartrate-resistant acid phosphatase; VET: vertical epidermal thickness.

5. Toxicology

Though some extracts or compounds from RRR have shown antitumor effects, rubiadin was reported to display carcinogenic potential. In the outer medulla, cytoplasmic swelling with basophilic changes and karyomegaly were observed in male F344 rats fed with rubiadin for one week whereas 26-week oral administration of the component induced atypical tubules, putative pre-neoplastic lesions, and karyomegaly. The results indicated that rubiadin may be a potent carcinogenic ingredient that targeted the proximal tubule cells in the outer medulla [96]. Rubiadin was also considered as both initiator and promoter of carcinogenicity targeting kidney, liver and large intestine [97]. In madder pigment, alizarin, purpurin and 1-hydroxyanthraquinone were found to have similar effects as ethidium bromide, a typical DNA intercalator. They exhibited potential genotoxicity by implanting into the DNA of Escherichia coli, blocking gene expression and inducing cell death [98].

6. Conclusions and Remarks on Future Work

As one of the earliest TCMs used in the clinic, RRR has shown various actions on many syndromes and indications for over 2000 years. In this paper, we have provided a review of RRR focused on the fields of botany, phytochemistry, pharmacology and toxicology based on the data and results collected from a large amount of research studies.

Rubidate is a synthetic derivative of ruberythric acid. It was documented to have the property of increasing leukocyte levels in peripheral blood [99,100] and developed into a medicine for the treatment of the leucopenia [101] in China in the 1980s. At present, over 100 components have been isolated from RRR, including anthraquinones, naphthoquinones, cyclic hexapeptides and triterpenoids, etc. In structure-effect relationship studies, with bioactivity-guided and high-throughput screening methods, other components from RRR or their synthetic derivatives may potentially be found as candidate drugs like rubidate.

In traditional clinical use, RRR has a long history of use in some hemorrhages induced by blood stasis for its activities of stopping bleeding and resolving stasis, which had been proved by some pharmacological experiments [74,75,76]. However, according to modern pharmacology, the two effects are contradictory on some indicators of coagulative and fibrinolytic systems. In a pathological state, stanching would cause stasis and blood-activating would induce hemorrhages, so there still remain some questions to be answered, like what are the active components or fractions responsible for these two seemingly conflicting functions, respectively? How can they act to maintain a healthy state of relative equilibrium rather than pathological state of hemorrhage or stasis?

There is no doubt that RRR is an effective TCM in clinical practice with a long history and plenty of accumulated experiences. Nevertheless, the mutagenicity or carcinogenic potential of the active anthraquinones is a matter of concern [102,103]. At present, there is lack of research and literature on the in vivo metabolism and the metabolites of these components, so in terms of safe medication, the Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) of these naturally occurring anthraquinones should be the urgent concern right now.

The present literature review provides a full-scale profile of various aspects of Rubiae Radix et Rhizoma and proposes some issues worth investigating in the future. We believe that it will help us to comprehensively understand and more effectively develop this traditional phytomedicine.