Introduction

Traditional Chinese medicine (TCM) is often considered as complementary to Western medicine in treating various types of cancer. It is a major part of the use of botanical medicine that is widespread in all regions of develo** world and is consistently growing in popularity in industrialized countries, especially among patients diagnosed with cancer [1]. Prunella vulgaris, a perennial herb, grows natively across East Asia throughout China, Japan, Korea, and Europe. It is commonly used as a dietary supplement [2]. As a folk medicine used for thousands of years in China, it has been mainly used as an antipyretic remedy for alleviating sore throat, reducing fever, and accelerating wound healing [3, 4•]. Its major bioactive ingredients consist of triterpenoids, steroids, flavonoids, coumarins, organic acid, volatile oil, phenylpropanoids, and carbohydrate [5]. Most of these substances have been shown a wide spectrum of biological properties including anti-viral [6, 7], anti-oxidant [8, 9], anti-microbial [10, 11], anti-inflammatory [12, 13], anti-diabetic [14, 15], anti-estrogenic [16], anti-allergic [17], immune modulatory [18, 19], and anti-cancer [20, 21] effects. In China, this well-known traditional herb is used as the key ingredient in many Chinese herbal mixtures in the forms of decoction, capsule, granule, or infusion for treatment of cancer, primarily as an adjuvant/complementary treatment in the majority of patients (see Table 1).

Table 1 Chinese herbal formula containing Prunella vulgaris
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Of these formulas containing P. vulgaris, anti-tumor-B (ATB) have shown strong cancer chemopreventive activities both preclinically and in clinical trials. In a clinical study [56], a total of 449 patients with esophageal epithelial hyperplasia were randomly divided into an anti-tumor B treatment (n = 300) and a placebo control group (n = 149). After 6 months of oral administration, the response rate of the ATB group was 64.4 vs 22.8 % compared to placebo group (p < 0.001) and the frequency of progression was 3.3 and 24.8 % (p < 0.001), respectively. In a 4-nitroquinoline-1-oxide (4NQO) induced oral squamous cell carcinoma model in A/J mice [55], ATB inhibited tumor development by 59.2 %. ATB treatment resulted in a significant reduction in multiplicity and tumor load in a A/J mutant lung adenocarcinoma model [57]. ATB inhibited the incidence of bladder cancer by 90.7 % (p < 0.01) in a rat bladder cancer model treated with N-butyl-(4-hydroxybutyl) nitrosamide (BBN) [58]. In general, ATB has been shown to be a potent cancer chemopreventive agent. However, it is still not clear if P. vulgaris is responsible for the observed chemopreventive efficacy of ATB or in any other Chinese herbal mixtures containing P. vulgaris. Future studies should focus on the role P. vulgaris in ATB’s efficacy as well as its chemopreventive effects as a single agent.

Chemical Constituents of P. vulgaris

P. vulgaris contains nine categories of chemical compounds isolated by various methods (listed in Tables 2 and 3). Four of the nine structural categories, namely, triterpenoid, flavonoid, phenylpropanoids, and steroids are found to have strong anti-tumor properties. Among these, triterpenoids have been the most widely investigated probably due to its high content in P. vulgaris. Kyun Lee et al. [94] isolated fifteen triterpenoids through the method of methanol extraction of P. vulgaris. Of the extracted triterpenoids, ursolic acid and oleanic acid are the two key constituents [59] that have shown anti-cancer properties (Tables 2 and 3). Ursolic acid [95] has anti-cancer effects against gallbladder cancer through suppression of proliferation, cell cycle arrest, and increased tumor cell apoptosis. Two new triterpenoids, pentacyclic triterpenoid [96] glycosides Vulgasides I and II have recently been isolated from P. vulgaris. The amount of flavonoid [97] was 2.2–10.3 % in P. vulgaris. Other phytochemicals, such as rutin, quercetin, and hyperoside, are found to have anti-cancer properties, including anti-proliferation, immune-enhancing, anti-oxidant, pro-apoptosis, cell cycle arrest in in vivo or in vitro studies. Phenolic acids consisting of rosmarinic acid and caffeic acid may also play a role in the anti-tumor properties of P. vulgaris through the mechanism of anti-angiogenesis, anti-proliferation, and induction on apoptosis. In the 7,12-dimethylbenz[a]anthracene (DMBA) induced skin tumor model using Swiss albino mice [88], oral administration of rosmarinic acid completely prevented the formation of skin tumors. Finally, five additional categories of chemical compounds are steroid, coumarins, organic oils, carbohydrates, and volatile oils. Steroid category [59] extracts include β-sitosterol, stigmasterol, and α-spinasterol.

Table 2 Key ingredients contained in Prunella vulgaris
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Table 3 Chemical structures of the major anti-cancer chemical compounds in Prunella vulgaris
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In Vitro Anti-cancer Activity (Table 3)

Anti-proliferation

Several studies [67, 71, 98–109] revealed that P. vulgaris inhibits the proliferation of human cancer cell lines (Table 3), including human esophageal cancer cell line Eca 109 [110, 111], liver cancer cell line HepG2, cervical cancer Hela cell, and stomach cancer MKN 45 cell line [8]. Possible mechanisms suggested include the inhibition of the c-Jun N-terminal kinase (JNK) pathway [1, 2] and the Akt pathway [2, 3]. Ethanol extract of P. vulgaris was found to inhibit colon cancer cell line HT-29 by arresting the cell cycle at the G1/S checkpoint and reducing the expression of pro-proliferative cyclin D1 and cyclin-dependent kinase 4 (CDK4) at the transcriptional and translational level [108]. P. vulgaris has also been shown to have combinatorial effects with other agents. For example, P. vulgaris extracts enhanced the effects of paclitaxel (TAX) and adriamycin (ADM) on inhibiting cell growth of cancer cells [105]. A combination of P. vulgaris and Cremastra appendiculata exhibited an enhanced effects in inhibiting the growth of thyroid cancer cell line [112, 113], along with down-regulation of the c-myc expression.

In addition to P. vulgaris extracts, some of its ingredients have also been examined for anti-cancer activities (Table 3). Triterpenoicacids, a component of P. vulgaris [94, 106, 107], exhibited strong cytotoxic activity against human lung cancer cell line A459. Triterpenoic acid [94] isolated from P. vulgaris has been shown to inhibit cell growths of various human cancer cell lines, namely, A549 cell lines, SK-OV-3 (ovarian cancer cell), SK-MEL-2 (skin melanoma), and HCT 15 (colon cancer cell). Ursolic acid [67], one of the most abundant triterpenoic acids in P. vulgaris, showed inhibitory effect on colon cancer cell lines HCT-15 and DLD-1. The mechanism [67] underlying ursolic acid-mediated anti-proliferation against human colon cancer cell lines is believed to be related to the N-terminal phosphorylation and subsequent proteasomal degradation of β-catenin. Ursolic acid also reduced proliferation in many other tumor cell lines, like human leukemic cell line HL-60 [114], mouse melanoma cell line B16 [115], human breast MCF7 [116]. Oleanic acid, an active component of P. vulgaris [71], inhibited the proliferation of HT-29 cells in dose-dependent manner through the mechanism of G0/G1 checkpoint arrest. Oleanolic acid [70] also exhibited strong anti-proliferation activity against human lung SPC-A-1 cells. Interestingly, an endophytic fungus CPCC 480171 [117] isolated from P. vulgaris was found to have cytotoxic effects on multiple human cell line, A549 (lung cancer), LOVO cells (colon cancer), CEM cells (T cell leukemia), and HL-60 (leukemia). Caffeic acid, a major phenolic compound in P. vulgaris [93], was observed to inhibit cancer cell proliferation, especially at a high concentration (over 30 μg/ml). Table 4 summarized in vitro efficacy of P. vulgaris and its extracts or components against cancer cell lines.

Table 4 In vitro anti-cancer activities
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Regulation of Cell Cycle Progression and Cell Cycle Arrest

P. vulgaris has been shown to induce cell cycle arrest at various checkpoints in cancer cells. After thyroid carcinoma cell line SW579 [109] was treated with P. vulgaris, the proportion of cells in the S phase was observed to be reduced, while those in the G0/G1 phase was significantly increased when compared to the control group. In another study [108], the ethanol extract of P. vulgaris arrested cells at the G1/S checkpoint in human colon carcinoma cells and inhibited the expression of both cyclin D1 and CDK4. Rutin [83], one of the flavonoids from P. vulgaris, showed anti-tumor effect against human neuroblastoma LAN-5 cells by inducing G2/M cell cycle arrest and apoptosis. Ursolic acid, another component of P. vulgaris, was shown to block B16 mouse melanoma cell line in G1 phase [115]. These reports suggest that P. vulgaris is capable of inducing cell cycle arrest in various cancer cell lines.

Induction of Apoptosis

Apoptosis [143–148] has been shown to be induced by many anti-tumor regimens [145–148] such as chemotherapy, radiation as well as Chinese herbs such as P. vulgaris. P. vulgaris and its components have been shown to induce apoptosis in a variety of cancer cell lines (including Raji cells [1, 4•], SGC-7901 [119], SW 579 [109, 112], Eca 109 [111], EL-4 [121], Jurket cells [103, 104], PANC-1 [122], T24 [136], HepG2, HT29, A549, MKN-45, and Hela cells [8]). Several phytochemicals from P. vulgaris including oleanic acid [70], ursolic acid [65], rosmarinic acid [126], and caffeic acid [93] have also been shown to either induce or promote apoptosis in cancer cells. Mechanisms suggested by several studies are both the up-regulation of the expression of p53 [65], Bax [8, 64, 70, 101, 104, 111, 122], Fas [136], Bad [70], caspase 3 [64, 118], and caspase 9 [64] as well as down-regulation of the expression of c-myc [119], Bcl-2 [64, 70, 101, 104, 121, 122, 149], Mcl [64], and Bcl-xl [64]. Other mechanisms have been suggested are the inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway [126], the mitochondrial pathway [150, 151], the nuclear transcription factor NF-κB [127] pathway, and the intracellular generation of reactive oxygen species (ROS) [61, 68, 77, 130, 131, 152, 153].

Anti-angiogenesis

P. vulgaris extracts [154] and rosmarinic acid [89] exhibited a dose-dependent inhibition of in vitro angiogenic properties of endothelial cells, including proliferation, metastasis, adhesion, and tube formation. Inhibition of ROS production was implicated as a potential mechanism for the decreased VEGF expression and decreased interleukin 8 (IL-8) release in response to rosmarinic acid treatment [89]. Triterpene acids [133], ursolic acid, and oleanolic acid, have been shown to inhibit angiogenesis in a dose-dependent manner in the chick chorioallantoic membrane (CAM) assay. Both ursolic acid and oleanic acid inhibited the proliferation of bovine arotic endothelial cells [133]. Jae HC et al. [137•] found that aqueous extract of P. vulgaris inhibited tumor angiogenesis and metastasis through suppression of NF-κB activation and phosphorylation of ERK1/2 by reducing the expression matrix metalloproteinase 9 (MMP-9). Aqueous extract of P. vulgaris, called PVAE [141], inhibited the migration and metastasis of human liver cancer cell lines by inhibiting the enzymatic activity and protein expression of MMP-2 and MMP-9.

In Vivo Anti-cancer Activity (Table 5)

Table 5 In vivo anti-cancer activities
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P. vulgaris has been characterized for its in vivo anti-cancer effects in multiple animal models (Table 5). Using Lewis lung carcinoma model, P. vulgaris [155•] has shown a significant anti-tumor effect when compared with control group. Using a human colon carcinoma HT-90 cell xenograft athymic nude mouse model [124], ethanol extract of P. vulgaris (also called EESP) significantly reduced tumor load when compared to control group without any signs of toxicity. In a T cell lymphoma EL-4 cell transplanted C57BL/6 mice model [20], ethanol extract of P. vulgaris inhibited tumor growth. In a benzo(a)pyrene intraperitoneal (i.p) injected A/J mice model [21], ethanol extract of P. vulgaris inhibited lung tumor multiplicity by more than 90 %; rosmarinic acid, a key component of P. vulgaris, has been shown to inhibit skin and colon carcinogenesis in a DMBA-induced skin carcinogenesis Swiss albino mice model [140] and in a rat model of colon carcinogenesis [92], respectively.

Clinical Studies (Table 6)

Table 6 Clinical studies
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Table 6 summarized clinical studies reported for P. vulgaris. Zhang et al. [157••] reported a randomized clinical trial (RCT) in which 101 patients with non-Hodgkin’s lymphoma were divided into three groups: EPVL (extract of P. vulgaris), CHOP (combined chemotherapy regimen), and EPVL + CHOP. CHOP consists of (C)yclophosphamide, (H)ydroxydaunorubicin, (O)ncovin (vincristine), and (P)rednisone or (P)rednisolone. A 70 % curative efficacy were observed in the combination group (EPVL + CHOP) comparing with 52.6 % in the chemotherapy group (CHOP) and 10.5 % in P. vulgaris alone (EPVL) group, indicating that P. vulgaris may serve as an effective adjuvant treatment with chemotherapy for non-Hodgkin’s lymphoma [157••]. Zhou RY [158] reported another randomized clinical trial of 23 late stage liver cancer patients into two groups: treatment group in which P. vulgaris (EESP-ethanol extract of Spica prunella) and a formulated injectable mixture called “anti-inflammation injection formulation #1” were perfused through liver artery; and control group with cisplatin (DDP), hydroxycamptothecin (HCPT), and 5-fluorouracil (5-FUu) perfused. Although there was no significant difference in the 1-year survival rates between the two groups, there is a significant difference of clinical symptoms with a higher Karnofsky Performance Scale (KPS) index (a higher KPS score indicates less functional impairment) was seen in the P. vulgaris group (p < 0.01). A similar observation was also seen in another study [159] in which P. vulgaris injection showed better results in controlling cancer-associated symptoms and higher KPS scores in patients with late stage gastrointestinal cancer. Zhou et al. [161••] reported the randomization of a group of 52 bronchopulmonary carcinoma patients with moderate or severe hydrothorax into two groups: one group received tube closed drainage along with intrapleural injection of P. vulgaris, and the control group received chemotherapy alone. The curative effect rate, average remission period, adverse reaction rate between P. vulgaris group, and chemotherapy alone group were 85 vs 46 %, 7 vs 1.5 months, and 0 ∼ 12 % vs 4∼ 35 %, respectively (p ≤ 0.05). This observation is confirmed by another study [160] on using P. vulgaris injection to treat hydrothorax in 78 patients as compared with the chemotherapy alone group. These results suggest that P. vulgaris is a potentially non-toxic therapeutic agent for the treatment of hydrothorax caused bronchopulmonary carcinoma.

Conclusion

P. vulgaris has been extensively used in China both independently and as a part of a multi-modal approach to treat cancer patients with standard chemotherapy. P. vulgaris appears to target multiple signaling pathways and has a complex mechanism of action. The complexity of the herb may be a key element of its therapeutic or preventive effectiveness. However, the pleiotropic effects that it causes make determining definitive targets for future pharmaceutical development more challenging. Based on its strong efficay in both pre-clincial model systems and in a number of clinical trials with limited toxicity or adverse effects, further studies should focus on characterizing P. vulgaris as a promising cancer chemopreventive agent.