The Hedyotis diffusa Willd. (Rubiaceae): A Review on Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics

Hedyotis diffusa Willd (H. diffusa) is a well-known Chinese medicine with a variety of activities, especially its anti-cancer effect in the clinic. Up to now, 171 compounds have been reported from H. diffusa, including 32 iridoids, 26 flavonoids, 24 anthraquinones, 26 phenolics and their derivatives, 50 volatile oils and 13 miscellaneous compounds. In vitro and in vivo studies show these phytochemicals and plant extracts to exhibit a range of pharmacological activities of anti-cancer, antioxidant, anti-inflammatory, anti-fibroblast, immunomodulatory and neuroprotective effects. Although a series of methods have been established for the quality control of H. diffusa, a feasible and reliable approach is still needed in consideration of its botanical origin, collecting time and bioactive effects. Meanwhile, more pharmacokinetics researches are needed to illustrate the characteristics of H. diffusa in vivo. The present review aims to provide up-to-date and comprehensive information on the phytochemistry, pharmacology, quality control and pharmacokinetic characteristics of H. diffusa for its clinical use and further development.


Introduction
Hedyotis diffusa Willd (H. diffusa, Family Rubiaceae), known as Oldenlandia diffusa (Willd) Roxb, is a well-known Chinese medicine used for the treatment of inflammation-linked diseases, such as hepatitis, appendicitis and urethritis, for thousands of years in China [1]. In our previous studies, the water extract of H. diffusa has been proved to have an obvious protective effect in lipopolysaccharide-induced renal inflammation in mice. Recently, H. diffusa has gained increasing attention for its properties of anti-proliferative activity in cancer cells and anti-tumor activity in tumor-bearing animals [2][3][4][5]. It has been proved as the most commonly prescribed single Chinese herb used for colon cancer and breast cancer patients [6,7], according to the statistics from the National Health Insurance Research Database of Taiwan.
H. diffusa is an annual herb, widely distributed in the orient and tropical Asia, such as China, Japan and Indonesia [1,8]. Generally, the plant grows in humid fields and ridges of farmlands, ascending to procumbent, to 50 cm tall; the stem is slightly flattened to terete, glabrescent to glabrous and the papilla was observed in the transverse section of the stem; the leaves are opposite, sessile or subsessile and blade drying membranous, linear, narrowly elliptic, 1-4ˆ0.1-0.4 cm; the flowers with pedicels are   [21,34] Athraquinones 63 2-Methyl-3-methoxy anthraquinone C 16

Iridoids and Triterpenes
Iridoids are one of the most important components in H. diffusa with various bioactivities, such as anti-oxidant, neuroprotective and anti-inflammatory effects [33,53]. Accompanied with the analysis of the NMR spectra of the pure compounds, the methods of tandem mass spectrometry (MS n ) and time-of-flight mass spectrometry (TOF/MS) have become more popular for the identification of these compounds [11,14,15,25,52]. To date, thirty-two iridoids and their iridoid glucosides (1-32) have been isolated and identified from H. diffusa (Figure 1).

Iridoids and Triterpenes
Iridoids are one of the most important components in H. diffusa with various bioactivities, such as anti-oxidant, neuroprotective and anti-inflammatory effects [33,53]. Accompanied with the analysis of the NMR spectra of the pure compounds, the methods of tandem mass spectrometry (MS n ) and time-of-flight mass spectrometry (TOF/MS) have become more popular for the identification of these compounds [11,14,15,25,52]. To date, thirty-two iridoids and their iridoid glucosides (1-32) have been isolated and identified from H. diffusa (Figure 1).

Flavonoids
Flavonoids are a major group presented in H. Diffusa, and most of them are derivatives of the flavonol aglycones of kaempferol and quercentin. Recently, other aglycones, such as chrysin, oroxylin and wogonin, have been characterized by ultra-performance liquid chromatography-diode array detector/quadrupole time-of-flight mass spectrometry (UPLC-DAD/Q-TOF-MS). To date, twenty-six flavonoids (37-62) with various substitutions have been identified and their chemical structures are prescribed in Figure 2.

Flavonoids
Flavonoids are a major group presented in H. Diffusa, and most of them are derivatives of the flavonol aglycones of kaempferol and quercentin. Recently, other aglycones, such as chrysin, oroxylin and wogonin, have been characterized by ultra-performance liquid chromatographydiode array detector/quadrupole time-of-flight mass spectrometry (UPLC-DAD/Q-TOF-MS). To date, twenty-six flavonoids (37-62) with various substitutions have been identified and their chemical structures are prescribed in Figure 2.

Anthraquinones
Anthraquinones are also a major group of bioactive components in H. diffusa. Up to now, twenty-four anthraquinones with various substitutions (63-86) have been obtained and identified from this herb. These compounds have a typical characteristic of the 9, 10-anthraquinone skeleton with the presence of hydroxy, methyl and/or methoxy groups, for example, 2-hydroxy-3-methoxy-6-methyl anthraquinone (77). Their chemical structures are shown in Figure 3.

Anthraquinones
Anthraquinones are also a major group of bioactive components in H. diffusa. Up to now, twenty-four anthraquinones with various substitutions (63-86) have been obtained and identified from this herb. These compounds have a typical characteristic of the 9, 10-anthraquinone skeleton with the presence of hydroxy, methyl and/or methoxy groups, for example, 2-hydroxy-3-methoxy-6methyl anthraquinone (77). Their chemical structures are shown in Figure 3.

Polysaccharides
The polysaccharides in H. diffusa have been researched for their immuno-enhancing activity. They are mainly composed of glucose, galactose and mannose, with the content of 15.10% determined by the spectrophotometry method at 490 nm [54]. Up to now, only one homogeneous polysaccharide, ODP-1, has been separated from H. diffusa, with the relative molecular weight of 20.88 kDa. It consists of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose, with the molar ratio of 0.005:0.033:0.575:1.000:0.144:0.143 [50].

Essential Oils
The reports of essential oils in this plant were mainly on isolating many fatty acids, fatty acid esters, etc. [11]. Yang et al. [49] identified 29 compounds representing 81.45% of the total oil content by GC/MS combined with the Kovats Retention index. n-Hexadecanoic acid (119) (31.22%), oleic acid (157) (6.74%), tetracosane (161) (4.94%) and 9,12-octadacadienoic acid (125) (4.87%) were found to be the main constituents. Liu et al. [48] compared the constituents and their content in H. diffusa collected from the provinces of Guangdong, Jiangxi and Guangxi in China and also revealed that the oil of H. diffusa was mainly composed of fatty acids with an oil extraction rate from 0.25% to 0.30%.

Polysaccharides
The polysaccharides in H. diffusa have been researched for their immuno-enhancing activity. They are mainly composed of glucose, galactose and mannose, with the content of 15.10% determined by the spectrophotometry method at 490 nm [54]. Up to now, only one homogeneous polysaccharide, ODP-1, has been separated from H. diffusa, with the relative molecular weight of 20.88 kDa. It consists of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose, with the molar ratio of 0.005:0.033:0.575:1.000:0.144:0.143 [50].

Essential Oils
The reports of essential oils in this plant were mainly on isolating many fatty acids, fatty acid esters, etc. [11]. Yang et al. [49] identified 29 compounds representing 81.45% of the total oil content by GC/MS combined with the Kovats Retention index. n-Hexadecanoic acid (119) (31.22%), oleic acid (157) (6.74%), tetracosane (161) (4.94%) and 9,12-octadacadienoic acid (125) (4.87%) were found to be the main constituents. Liu et al. [48] compared the constituents and their content in H. diffusa collected from the provinces of Guangdong, Jiangxi and Guangxi in China and also revealed that the oil of H. diffusa was mainly composed of fatty acids with an oil extraction rate from 0.25% to 0.30%.

Pharmacology
H. diffusa has long been used therapeutically in China, due to its broad spectrum of biological and pharmacological activities. Now we have enlisted an overview of the modern pharmacological studies in the following sections (Table 2).

Pharmacology
H. diffusa has long been used therapeutically in China, due to its broad spectrum of biological and pharmacological activities. Now we have enlisted an overview of the modern pharmacological studies in the following sections (Table 2). HT-29 cells Ethanol extract The extract reduced HT-29 cell viability and survival. It could suppress cancer cell proliferation by blocking the cell cycle, preventing G 1 to S progression, and reducing mRNA expression of pro-proliferative PCNA, Cyclin D1 and CDK4, but increasing that of anti-proliferative p21. [55]

HT-29 cells Ethanol extract
The extract induced the HT-29 cell morphological changes and reduced cell viability. In addition, the extract treatment resulted in DNA fragmentation, loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. [56] HT-29 cells Ethanol extract The extract treatment downregulated the mRNA and protein expression levels of VEGF-A in HT-29 human colon carcinoma cells. [57] HT-29 cells Ethanol extract The extract inhibits colorectal cancer growth in vivo via inhibition of SHH-mediated tumor angiogenesis. [58] CRC mouse xenograft model Ethanol extract The extract inhibited the expression of the gene VEGF-A and VEGFR2, thus, suppressed the activation of Sonic hedgehog (SHH)-signaling in CRC xenograft tumors; it inhibits colorectal cancer growth. [58] CRC mouse xenograft model Ethanol extract The extract suppressed the STAT3 pathway by suppressing STAT3 phosphorylation in tumor tissues, altering the expression pattern of target genes of Cyclin D1, CDK4 and Bcl-2, as well as upregulating p21 and Bax. Leukemia

CEM cells Aqueous extract
The extract inhibited Leukemia CEM cells growth in time-and concentration-dependent manners. And the inhibition mechanism has greater correlation with the upregulation of P53 expression. The extract could induce HL-60 cells differentiation, and suppress the expression of the anti-apoptosis-related gene to inhibit the growth of HL-60 cells. [65]

HL-60 cells, WEHI-3 cells Ethanol extract
The extract inhibited the cell proliferation of HL-60 cells. It triggered an arrest of HL-60 cells at the G 0 /G 1 phase and sub-G 1 population, provoked DNA condensation and DNA damage, but the activities of caspase-3, caspase-8, and caspase-9 were elevated in H. diffusa-treated HL-60 cells. [66] U937 cells 2-Hydroxy-3-methyl anthraquinone 2-Hydroxy-3-methyl anthraquinone enhanced apoptosis of U937 cells through the activation of p-p38MAPK and downregulation of p-ERK1/2. [67] THP-1 Cells 2-Hydroxy-3-methyl anthraquinone 2-Hydroxy-3-methyl anthraquinone induced THP-1 cell apoptosis, which was associated with a more prominent induction expression of Fas/FasL, DR4 and TRAIL. Moreover, 2-Hydroxy-3-methylanthraquinone treatment resulted in activation of caspase-8. [68] Liver cancer H22 mice Aqueous extract The extract had an inhibitory effect on the metastasis of hepatocarcinoma in blood. [69] HepG2 cells Aqueous extract The extract remarkably inhibited HepG2 cell proliferation via arrest of HepG2 cells at the G 0 /G 1 phase and induction of S phase delay. In addition, the extract potentiated the anticancer effect of low-dose 5-FU in the absence of overt toxicity by downregulating the mRNA and protein levels of CDK2, cyclin E and E2F1. [70] MHCC97-H cells Total flavones extract The extract treatment reduced the level of E-cadherin protein and increased the expression of vimentin protein in TGF-β1-induced MHCC97-H. [71] HepG2 cells 1,3-Dihydroxy-2-Methylanthraquinone Ethyl acetate extract Both 1,3-Dihydroxy-2-Methylanthraquinone and ethyl acetate extract exhibited an inhibitory effect on HepG2 cells, resulting in in upregulation of Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C levels and caspase-3, -8, -9 proteases activities, while downregulating Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 in a dose-dependent manner. [72] HepG2 cells Nine pure compounds isolated from H. diffusa Ursolic acid exhibited a strong inhibition of cell survival with C 50 37 mM. [5] HepG2 cells 2-Hydroxy-3-methyl anthraquinone 1-Methoxy-2-hydroxy anthraquinone Both compounds showed inhibitory activity against protein tyrosine kinases v-src and pp60src and arrested the growth of HepG2 cancer cells. [38]  The extract suppressed the cell proliferation of A549 and H1355 cells as well as reduced cell viability in a concentration-dependent manner. [66]

Bcap37 cells 2-Hydroxy-3-methyl anthraquinone, 1-Methoxy-2-hydroxy anthraquinone
Both compounds showed inhibitory activity against protein tyrosine kinases v-src and pp60src and arrested the growth of Bcap37 cells. [38] Cervical tumor Nude mouse model Aqueous extract The extract had an inhibitory effect on cervical cancer cells with the expression of Ki-67 protein significantly decreased, and the mean survival time of the mice was significantly extended. [3] HeLa cells Nine pure compounds isolated from H. diffusa 2-Hydroxymethy-1-hydroxy anthraquinone exhibited the strongest inhibitory effect on cell viability. [5] Prostate Cancer

Immunomodulatory effect
Normal BALB/c mice Ethanol Extract The extract has promoted immune responses in normal BALB/c mice. [82] Immunosuppression mice induced by cyclophosphamide Polysaccharides extracts The extract could improve the clearance index, phagocytic index, and the index of the thymus and spleen of immunosuppression mice. [50] Inmmunosuppressed mice induced by cyclophosphamide Total flavonoids extract The extract enhanced specific and non-specific immunity. [83]

Antioxidant effects
The extract from methanol, acetone and 80% alcohol The extraction with 80% alcohol has the strongest antioxidant activity on DPPH assay. [84] The extract from water, ethanol, acetone, chloroform, ether, petroleum benzine Acetone extract had the strongest antioxidant effect. [ Twelve pure compounds isolated from H. diffusa All compounds showed antioxidant effects on xanthine oxidase inhibition, xanthine-xanthine oxidase cytochrome c and TBA-MDA systems. [33]

Anti-inflammatory effect
Lipopolysaccharide-induced renal inflammation mice Aqueous extract The extract protected renal tissues, significantly suppressed the production of TNF-α, IL-1, IL-6 and MCP-1, as well as significantly promoted the production of IL-10 in serum and renal tissues. [87]

RAW 264.7 cells Total flavonoids extract
The extract treatment on LPS-stimulated RAW 264.7 cells, reduced expression of iNOS, TNF-α, IL-6 and IL-1β, as well as suppressing phosphorylation of IκB p38, JNK and ERK1/2 in a concentration-dependent manner, indicating that the anti-inflammatory activity of total flavonoids had a close relationship with the NF-κB and MAPK signaling pathways. [88]

Rat cortical cells damaged by L-glutamate
Methanolic extract, five flavonoids and four O-acylated iridoid glycosides All compounds exhibited significant neuroprotective activity in primary cultures of rat cortical cells damaged by L-glutamate. [34] Anti-fibrosis effect

Ras oncogene-transformed R6 cells Oleanolic acid
Oleanolic acid inhibits the growth of ras oncogene-transformed R6 cells.
Oleanolic acid-mediated growth inhibition of transformed cells does not require direct cell-cell contact between normal and ras-transformed cells.

Anti-Colorectal Cancer Activity
H. diffusa has been used as a major formula for the clinical treatment of colorectal cancer (CRC). In vitro, ethanol extract of H. diffusa treatment significantly suppresses proliferation and induced apoptosis of HT-29 cells, resulting in DNA fragmentation, loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, reduction of the mRNA expression levels of cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma-2 (Bcl-2), upregulation of the expression levels of Bcl-2-associated X protein, prevention of G1-S progression, and reduction of mRNA expression of pro-proliferative PCNA, Cyclin D1 and CDK4. These results indicated that the anti-colorectal cancer cells effect of H. diffusa might be carried out via multiple approaches, such as the mitochondria-dependent pathway, IL-6/STAT3 pathway and cell cycle arrest [2,[55][56][57][58]. The mechanism was also confirmed by animal experiments [58,59]. Meanwhile, the ethanolic extract of H. diffusa displayed an inhibition effect on CT-26 cells with inhibitory rates from 35.46%˘3.59% to 71.84%˘3.12% at different concentrations (0.06 mg/mL, 0.08 mg/mL, 0.10 mg/mL, 0.12 mg/mL, 0.14 mg/mL, 0.16 mg/mL, 0.18 mg/mL and 0.20 mg/mL) and showed a stronger inhibition effect with an increase of concentration [60]. Li et al. [61] revealed that the ethanolic extract treatment could overcome 5-fluorouracil resistance in HCT-8/5-FU cells by downregulating the expression of P-gp and ABCG2. In addition, 2-hydroxymethy-1-hydroxy anthraquinone (IC 50 45 µM) and ursolic acid (IC 50 71 µM)) isolated from H. diffusa exhibited inhibition effects on Caco-2 cell proliferation [5], and the mechanism of the inhibition effect for ursolic acid might include the cleavage of the Poly (ADP-ribose) Polymerase (PARP) [62].

Anti-Leukemia Activity
The anti-leukemia effects of both aqueous and ethanolic extracts of H. diffusa have been investigated in several cancer cell lines. H. diffusa aqueous extract treatment with 0.01-4150 µg/mL restrained the growth of the CEM cells by enhancing the expression of P53 in vitro [63] and influenced murine leukemia WEHI-3 cells, as well as promoting T-and B-cell proliferation in leukemic mice administrated with 16 and 32 mg/kg in vivo [64]. The ethanolic extract of H. diffusa could trigger an arrest of HL-60 cells at the G 0 /G 1 phase and sub-G 1 population, provoke DNA condensation and DNA damage, but elevate the activities of caspase-3, caspase-8 and caspase-9, thus, inhibiting the cell proliferation of HL-60 cells with the half maximal inhibitory concentration (IC 50 ) value of 4.62 mg/mL [65,66]. Wang et al. [67] found that 2-hydroxy-3-methyl anthraquinone treatment (0-80 µM) could enhance apoptosis of U937 cells in a dose-dependent manner through the activation of p-p38MAPK and downregulation of p-ERK1/2. Further study verified it could alter the expression of Fas/FasL and activation of caspase-8, thus inducing THP-1 cell apoptosis [68].

Anti-Lung Cancer Activity
Aqueous extract of H. diffusa treatment (0-200 µg/mL) showed a suppression effect on A549 and H1355 cells in a concentration-dependent manner. But this effect was not found in LLC cells [66]. Further, Shi et al. [38] confirmed that two compounds of 2-hydroxy-3-methyl anthraquinone (IC 50 66 µM) and 2-hydroxy-1-methoxy anthraquinone (IC 50 79 µM) from H. diffusa could induce apoptosis on SPC-1-A cells with a close relationship to the mitochondrial apoptotic pathway.

Anti-Cervical Tumor Activity
Zhang et al. [3] discovered that the aqueous extract of H. diffusa treated (0.5 g/kg bw) by intragastric administration on human cervical carcinoma xenograft in nude mice showed an inhibitory effect on cervical cancer cells and induced apoptosis of Hela cells. Meanwhile, anthraquinones, especially 2-hydroxymethy-1-hydroxy anthraquinone, showed a strong inhibitory effect on Hela cells with IC 50 45 µM in vitro [5].

Anti-Prostate Cancer Activity
The potential anti-prostate cancer effect of H. diffusa, mainly the active compounds, has previously been provided on a variety of cell lines. 2-Methyl-3-methoxy anthraquinone (IC 50  Hu et al. [51] isolated three cyclotides (DC 1-3) and studied their anti-prostate cancer effect. Thus, three cyclotides, especially DC 3 (1 mg/kg) showed inhibition against PC3, DU145 and LNCap cells. In addition, DC3 significantly inhibited development of the tumor in weight and size in the model of a prostate xenograft, and showed significant anti-cancer effect (p < 0.01) at a dose of 1 mg/kg, with 40.23% inhibition of the rate of tumor growth (weight).

Other Anti-Cancer Effects
Other anti-cancer effects have also been reported during these years. The ethanolic extract of H. diffusa (0-200 µg/mL) suppressed the proliferation of B16F10 cells in a dose-dependent manner [66]. The lipophilic extract (50 and 100 mg/kg) and crude polysaccharide (31.25 and 62.5 mg/kg) from H. diffusa showed anti-tumor activities on S-180 cells and a protective effect on chemotherapeutic damage [77]. H. diffusa injection could induce the apoptosis of MG-63 cells by increasing the Bax gene expression in a concentration-dependent manner from 50 to 400 µg/mL [78,79]. When it is used with cisplatin, the combined use exhibited a stronger inhibitory effect than the single agents. This might be due to its property of elevating the levels of Bax, Bad, caspase-3 and caspase-8 expression and decreasing the levels of Bcl-xl and Bcl-2 [80]. Meanwhile, Zhang et al. [4] found that the aqueous extract of H. diffusa (2-8 mg/mL) inhibited the growth of U87 cells in a dose-dependent manner by inducing mitochondrial apoptosis via the AKT/ERK pathways. Moreover, the compound, 4-vinyl phenol, was demonstrated to have anti-angiogenic activity in human endothelial cells of HUVEC (IC 50 15.31 µg/mL) and HMEC -1 (IC 50 21.43 µg/mL), breast tumor-bearing BALB/c mice (0.2-2 mg/kg), C57BL/6 mice (20-100 µg/mL matrigel) and zebrafish embryo models (6.25-12.5 µg/mL matrigel), and this effect had a close relationship with the PI3K/AKT pathway [81].

Immunomodulatory Effect
Lin et al. [64] found that aqueous extract of H. diffusa (16 and 32 mg/kg) affected immune responses by promoting T-and B-cell proliferation in leukemic mice in WEHI-3-generated leukemia mice. Meanwhile, Kuo et al. [82] discovered that the ethanolic extract (16, 32 and 64 mg/kg) could also promote immune responses in normal BALB/c mice by promoting CD11b, CD19 and Mac-3 levels, increasing phagocytosis activity of macrophages obtained from the peritoneal cavity and increasing NK cell activity and B-and T-cell proliferation. The polysaccharides extracts (2.25, 4.5 and 9.0 mg/mL) could improve the clearance index, phagocytic index and the index of the thymus and spleen of immunosuppression mice [50]. When inmmunosuppressed mice were orally administrated total flavonoids of H. diffusa (15, 30 and 60 mg/kg), the levels of interleukin-2 (IL-2) and interferon-γ (INF-γ) were enhanced and the proliferation of T and B lymphocytes was increased, indicating the immunomodulatory effect of total flavonoids [83].

Anti-Inflammatory Effect
The aqueous extract (5.0 g/kg bw) treatment exhibited an anti-inflammatory effect in lipopolysaccharide-induced renal inflammation of mice by significantly suppressing the production of tumor necrosis factor-α (TNF-α), IL-1, IL-6 and monocyte chemotactic protein 1 (MCP-1) in renal tissues, as well as significantly promoting the production of IL-10 in serum and renal tissues. Moreover, two main chemotypes, including eight flavonoids and four iridoid glycosides were found in renal tissues after H. diffusa treatment, indicating that the anti-inflammatory effect may be due to these constituents [87]. In vitro, Chen et al. found that the flavonoids extract treatment (50´100 µg/mL) on LPS-stimulated RAW 264.7 cells reduced expression of iNOS, TNF-α, IL-6 and IL-1β, as well as suppressing phosphorylation of IκB p38, JNK and ERK1/2 in a concentration-dependent manner, indicating that the anti-inflammatory activity of total flavonoids had a close relationship with the NF-κB-and MAPK-signaling pathways [88]. isolated from H. diffusa exhibited a significant neuroprotective effect on L-glutamate-damaged rat cortical cells in the concentration from 0.1 to 10 µM; further, the structure-activity study proved di-OH in the B ring and an acyl substituent in flavonoids, a p-methoxy group in the aromatic ring and a trans double bond in the acyl moiety of acylated iridoid glycosides might be crucial for the biological response [34]. Wu et al. [89] found the inhibitory effect of oleanolic acid (2 and 8 µg/mL) isolated from H. diffusa against ras-transformed fibroblasts on R6 cells, and this inhibition might cause normal cells to secrete an inhibitory factor against the transformed cells, but did not require direct cell-cell contact.

Quality Control
Quality control of herbal medicines is necessary to ensure their stability, efficiency and safety. Modern analytical techniques provide simpler, more accurate and reliable methods for the quality control for H. diffusa. Besides the macroscopic and microscopic characters of H. diffusa [9], DNA sequence has become a powerful tool for the distinguishing H. diffusa from counterfeits, such as H. corymbosa and H. tenelliflora [8,12]. Chemical fingerprint is a comprehensive method accepted by the Food and Drug Administration, European Medicines Agency, and China Food and Drug Administration [90]. It can provide information about the types of compounds, as well as their relative ratios. A HPLC-MS fingerprint method was applied to 10 batches of H. diffusa materials from nine regions in China. The results showed that this method could differentiate samples from different geographical origins or processing methods [91]. Liang et al. [92] analyzed the chemical fingerprints of 17 batches of H. diffusa and found that the contents of asperuloside and (E)-6-O-p-coumaroyl scandoside methyl ester were quite different in samples collected from different habitats. Ursolic acid Oleanolic acid HPLC The contents of ursolic acid and oleanolic acid were 0.51%-0.58% and 0.11%-0.14%, respectively. And the contents of the whole herb were slightly lower than those of the overground part for both of the two compounds. [102] Ursolic acid Oleanolic acid HPLC-MS/MS The contents of ursolic acid and oleanolic acid for 10 batches were 0.15%-0.65% and 0.06%-0.17%, respectively.     The quantitative analysis for the quality control of H. diffusa has mostly focused on the diversity of components by a series of analytical methods, such as UV, HPLC, TLC and LC/MS. Up to now, triterpenes (ursolic acidand oleanulic acid), iridoids ((E)-6-O-p-coumaroyl scandoside methyl ester), geniposidic acid, deacetyl asperulosidic acid methyl ester, asperuloside acid, asperulosideand (E)-6-O-feruloyl scandoside methyl ester), phenolic acid (p-coumaric acid and ferulic acid), flavonoids (quercetin, rutin, quercetin-3-O-β-D-glucopyranside, quercetin-3-O-sambubioside, kaempferol, kaempferol-3-O-β-D-glucopyranside), anthraquinones (2-hydroxy-3-methoxy-7-methyl anthraquinone and 2-hydroxy-1-methoxy anthraquinone), polysassharides and one miscellaneous compound have been quantified as mark compounds for the quality control of H. diffusa. However, there were wide variations in the contents of these compounds, caused by samples from different sources and different collecting times (Table 3). Therefore, it is very urgent that a comprehensive method for ensuring the quality of H. diffusa be established.

Pharmacokinetics
The investigations about pharmacokinetics of H. diffusa are very scarce. After oral administration of H. diffusa in lipopolysaccharide-induced renal inflammation in mice, most compounds, including flavonoids, iridoid glycosides and anthraquinone, were found in plasma, and 12 compounds (eight flavonoids and four iridoid glycosides) were found in kidney, determined by UPLC-Q-TOF-MS/MS. The results indicated that flavonoids, iridoids and anthraquinones might be responsible for the protective effect of H. diffusa on renal inflammation [87]. Liu et al. [111] found that p-coumaric acid was a major metabolite of (E)-6-O-p-coumaroyl scandoside methyl ester in rat plasma after oral administration of a dose of 20 mg/kg. Compared with direct administration of p-coumaric acid, the absorption and elimination of p-coumaric acid were slower with administration of (E)-6-O-p-coumaroyl scandoside methyl ester. This was also confirmed by Yan et al. at 2011 [112]. Moreover, Ganbold et al. [62] investigated the bioavailability of H. diffusa by production of post-absorption samples using the Caco-2 cell model and confirmed that the decoction has good permeability (Papp = 3.575ˆ10´6 cm/s) in vitro with no cytotoxic effect.

Conclusions
Although H. diffusa has been used in China for thousands of years as a heat-clearing and detoxifying medicine, it has become popular for its anti-cancer effect, especially in the Taiwan district. Modern research on H. diffusa has provided much evidence for its anti-cancer effect using in vitro and in vivo experiments and has tried to clarify the mechanism of its action. Meanwhile, its other activities, such as anti-oxidant, anti-inflammatory, anti-fibroblasts, immunomodulatory and neuroprotective effects, have been reported. The achievement of these therapeutic effects is due to the chemical composition of H. diffusa. One hundred and seventy-one compounds have been reported, including iridoids, flavonoids, anthraquinones, phenolic acids and their derivatives, sterols, triterpenes, polysaccharides, cyclotides, coumarins, alkaloids and volatile oils. Among these constituents, iridoids, flavonoids and anthraquinones are three main ingredients and may play an essential role in its activities. However, there is no official quality standard for the quality control of H. diffusa. The contents of bioactive compounds are significantly different in the samples from different sources and different collecting times. So, a feasible and reliable approach is urgently needed in considering the botanical origin and bioactive effects. Moreover, a relatively small number of pharmacokinetics studies have been summarized, and, therefore, it is difficult to evaluate the function of H. diffusa in the human body. Altogether, this review gives comprehensive information about H. diffusa and provides evidence for its clinical application and further development.