Research Progress on Pharmacological Effects of Ardisia gigantifolia Stapf, a Traditional Chinese Medicine with Lingnan Characteristics

Weibo DAI, Qing LUO, Weijie PENG, Weiwen PENG

Zhongshan Hospital of Traditional Chinese Medicine, Affiliated to Guangzhou University of Chinese Medicine, Zhongshan 528401, China

Abstract This paper comprehensively reviews the research progress on the pharmacological effects of Ardisia gigantifolia Stapf in recent years, and finds that Ardisia gigantifolia Stapf has good pharmacological effects in many aspects, including anti-inflammation, anti-oxidation, anti-thrombosis, anti-tumor and so on. In particular, more than ten saponins have been found to have good anti-tumor activity and have potential research value, which can provide reference for the full development and utilization of Ardisia gigantifolia Stapf resources.

Key words Ardisia gigantifolia Stapf, Anti-tumor, Pharmacological effects, Research progress

1 Introduction

ArdisiagigantifoliaStapf is a medicinal plant in the Myrsinaceae family, and is distributed in Guangdong, Guangxi, Yunnan, Jiangxi and other places in China. It was first published inEssentialsofHerbalMedicinein the Qing Dynasty[1]. It has the effect of expelling wind and dehumidification, promoting blood circulation and removing blood stasis, and can be used for injuries from falls, rheumatism, arthralgia and so on[2]. In modern clinic, it is used in the treatment of gouty arthritis[3], rheumatoid arthritis[4], hyperosteogeny[5], bone fracture[6]and so on. The study of chemical composition shows thatA.gigantifoliaStapf contains phenols, quinones, sterols, triterpenoids and volatile oils, which has good pharmacological effects in anti-thrombosis, antioxidation, anti-tumor, anti-rheumatoid and so on. It is necessary to summarize the pharmacological activity comprehensively and deeply. In this paper, the pharmacological research literature ofA.gigantifoliaStapf in recent years was reviewed in order to provide a reference for the further development and utilization ofA.gigantifoliaStapf resources.

2 Pharmacological effects

2.1 Anti-inflammatory effectA.gigantifoliaStapf can play a role in the fight against rheumatoid arthritis by reducing the level of inflammatory factors and improving inflammatory swelling. Dai Weiboetal.[7-8]established a rat model of adjuvant-induced arthritis (AA) induced by Complete Freund’s Adjuvant (CFA), and observed the improvement effect of ethanol extract ofA.gigantifoliaStapf on the model animals. The results showed that the ethanol extract ofA.gigantifoliaStapf could significantly reduce the systemic and arthritis swelling scores of AA rats, reduce the joint swelling of AA model rats, reduce the systemic inflammation score of model animals, reduce the degree of foot swelling of inflammatory side and secondary side, and reduce the levels of IL-6, TNF-α and IL-1β in serum. It also significantly inhibited the infiltration of inflammatory cells, synovial hyperplasia and pannus formation in ankle joint tissue, and reduced the degree of cartilage and bone injury. This showed thatA.gigantifoliaStapf could improve the inflammatory state of AA model, reduce the level of inflammatory factors, improve the inflammation of ankle joint, and had a better effect in resisting rheumatoid arthritis. Further study on petroleum ether extract ofA.gigantifoliaStapf showed that it could significantly reduce the scores of systemic inflammation and arthritis swelling degree of AA rats, improve the histopathology of ankle joint, reduce the swelling degree of paws, reduce the content of serum MDA and PGE2 in inflammatory foot tissue, and reduce the organ index of thymus, spleen and liver. It suggested that petroleum ether extract fromA.gigantifoliaStapf may play a role in anti-rheumatoid arthritis by improving inflammatory state and ankle tissue lesion, inhibiting immune response, reducing oxidative damage and down-regulating the expression of inflammatory mediator PGE2 in AA rats.

2.2 Antioxidant effectThe antioxidant effect ofA.gigantifoliaStapf is mainly characterized by reducing the content of NO and malonaldehyde (MDA) and increasing the activity of catalase (CAT) and superoxide dismutase (SOD)invivo. Several compounds can inhibit the release of NO and increase the scavenging rate of 1, 1-Diphenyl-2-picrylhydrazyl radical (DPPH)invitro. Through the establishment of rat and rabbit thrombus models, Shen Shijunetal.[10]found that 60% alcohol extract ofA.gigantifoliaStapf could reduce the content of NO, increase the activity of CAT and SOD, reduce the content of MDA, inhibit lipid peroxidation and play an antioxidant role.

Yang Zhuetal.[11]tested the NO release activity of rat macrophages with ethyl acetate isolated from 60% alcohol extract ofA.gigantifoliaStapf. The results showed that gallic acid, bergenin and (-)-epicatechin had weak inhibitory effect at the tested concentration. Among the four phenolic anhydrides, Compound (-)-4′-hydroxy-3′, 5′-dimethoxyphenyl-β-D-[6-O-(4 -hydroxy-3″, 5′-dimethoxybenzoyl)]-glucoside and (-)-4′-hydroxy-2′, 6′-methoxyphenyl-β-D-[6-O-(4′-hydroxy-3″-methoxybenzoyl)]-glucoside had the strongest inhibitory effect on NO release, while Compound (-)-4′-hydroxy-3′-methoxyphenyl-β-D-[6-O-(4′-hydroxy-3″, 5′-dimethoxybenzoyl)]-glucoside and (-)-3′-hydroxy-4′-methoxyphenyl-β-D-[6-O-(4′-hydroxy-3″, 5′-dimethoxybenzoyl)]-glucoside had the weakest activity. The DPPH scavenging activity was testedinvitro, and the results showed that at the concentration of 100 μM, gallic acid, (+)-5-(1, 2-dihydroxypentyl)-benzene-1, 3-diol, (-)-epicatechin, (-)-4′-hydroxy-3′, 5′-dimethoxyphenyl-β-D-[6-O-(4″-hydroxy-3″, 5′-dimethoxybenzoyl)]-glucoside, (-)-3′-hydroxy-4′-methoxyphenyl-β-D-[6-O-(4″-hydroxy-3″, 5″-dimethoxybenzoyl)]-glucoside all exhibited good scavenging effect on free radicals, and the scavenging rate was higher than 80%.

Three bergenin derivatives[11-O-(3′-O-method-lgalloyl) bergenin, 11-O-galloylbergenin, 4-O-galloylbergenin]isolated from the rhizomes by Mu Lihuaetal.[12]had significant DPPH scavenging activity, and their half effective concentrations (EC50) were 9.7, 9.0 and 7.8 μmol/L, respectively.

2.3 Antithrombotic effectThe antithrombotic effect ofA.gigantifoliaStapf is mainly improving thrombosis, regulating lipid metabolism and improving microcirculation. Shen Shijunetal.studied the antithrombotic effect of alcohol extract ofA.gigantifoliaStapf, which showed that prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were prolonged, coagulation factor V and VI activity, whole blood viscosity and plasma fibrinogen (Fg) content were decreased, and endogenous and exogenous coagulation process was inhibited, so as to prevent thrombosis and reduce lung tissue injury. And the extract ofA.gigantifoliaStapf can reduce the content of MDA, increase the content of NO, enhance the activity of CAT and SOD, inhibit lipid peroxidation and exert the effect of antioxidation, so as to stabilize vascular endothelial cells and regulate lipid metabolism. The extract ofA.gigantifoliaStapf can also dilate capillaries, increase the number of open capillaries, accelerate the flow rate and flow pattern of red blood cells, improve microcirculation, and thus inhibit thrombosis in the body[13].

The studies of Liu Yanfangetal.have shown thatA.gigantifoliaStapf polysaccharides can prolong PT, APTT, TT and plasma recalcification time (RT), reduce plasma fibrinogen (Fg) and hemoglobin concentration, reduce hematocrit, inhibit endogenous and exogenous coagulation, improve blood viscosity, so as to prevent thrombosis and reduce lung tissue injury[14].

2.4 Anti-tumor effectTriterpenoid saponins are the active substances in the anti-tumor effect ofA.gigantifoliaStapf, and more than ten triterpenoids have been found to have good anti-tumor activity. Zhang Xiaoming[15]screened the anti-tumor activity of 218 species of medicinal plants in Guangxi with Hela cells by MTT method. It was found that the inhibition rate of 60% ethanol extract ofA.gigantifoliaStapf on Hela cells was 81.4% at the concentration of 200 μg/mL. Eleven triterpenoid saponins were isolated fromA.gigantifoliaStapf by anti-tumor and blood circulation tracing. Their antitumor activity was tested by four cell lines NCI-H 460, SF-268, MCF-7 and HepG2. Seven triterpenoid saponins in compounds 34-39 and 51 showed good antitumor activity. It suggested that the antitumor activity was related to the formation of glucose esters at position 6, and the formation of double bonds at position 12 and 13 might also play a major role in the antitumor activity.

Mu Lihuaetal.[16]put D 101 macroporous resin on 60% ethanol extract ofA.gigantifoliaStapf, and five triterpenoid saponins were isolated from 70% ethanol elution, TheIC50of 3β-O-{α-L-pyranosyl-(1 → 3)-[β-D-xyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-α-L-arabinosyl}-Siclamine A (AG4) on tumor cells BCG-823, EJ and HepG2 were 0.29, 9.99 and 2.03 μg/mL, respectively. TheIC50of lysikoianoside on tumor cells EJ was 7.20 μg/mL, and theIC50of 3β-O-{α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranose-(1→2)]-β-D-glucop-yranosyl-(1→4)-α-L-ara-bino-pyranosyl}-3β-hydroxy-13β, 28-epox-yoleanan-16-oxo-30-a hadIC50of 8.53 μg/mL for HepG2.

Gu Yongjie[17-18]found that AG4 also had strong inhibitory effect on tumor cells Bel-7402, HepG2 and SMMC-7721, and 3β-O-{α-L-pyranosyl-(1 → 3)-[β-D-glucopyranosyl-(1 → 3)-β-D-xyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 4)-[β-D-glucopyranosyl-(1 → 2)]-α-L-pyranoarabinosyl}-16, 28, 30-trihydroxy-oleanan-12-ene was sensitive to lung cancer cell line A549, andIC50of 24 h was 4.55 μmol/L. The mechanism of inducing apoptosis of A549 was related to blocking G2 phase and increasing S phase. The effect of biotransformation product S1 ofA.gigantifoliaStapf on 6 tumor lines including Bel-7402, MCF-7 and A549 was studied. It was found that the range ofIC50was 12.8-27.3 μmol/L, and Bel-7402 cell line was the most sensitive to S1. After S1 interfered with Bel-7402 cells for 24 h, the early apoptosis was obvious, and the apoptosis rate was obviously concentration-dependent. The cells were blocked in G2/M phase, and the cells in this phase increased obviously, while the cells in S phase decreased obviously. The anti-tumor activity was achieved by inhibiting cell proliferation. This shows that the biotransformation product S1 ofA.gigantifoliaStapf has a certain inhibitory effect on six kinds of tumor cell lines, and has a strong inhibitory effect on Bel-7402 cells. The mechanism of inducing apoptosis of Bel-7402 cells may be related to the arrest of cell cycle[19].

Chen Chaoetal.[20-21]studied the effect of AG4 on the transplanted tumor of CNE nude mice. It was found that AG4 could significantly inhibit the tumor weight and volume of the transplanted tumor and inhibit its growth. The mechanism is related to the activation of mitochondrial pathway to induce tumor cell apoptosis, promote Bax and Bad gene expression and inhibit Bcl-2 gene expression.

Zheng Xiaolietal.[22]showed that AG4 had a strong inhibitory effect on human breast cancer cell line MCF-7, and the 24 hIC50was 3.67 μmol/L. AG4 can increase the S phase cells and decrease the G2/M phase cells. It has the effect of cycle arrest. AG4 can decrease the SOD activity and GSH content in MCF-7 cells, increase the MDA content, and interfere with the redox system in MCF-7 cells.

He Shanetal.[23-25]found that the expression of DUSP1, DUSP4, DUSP5 mRNA and protein in hepatoma HepG2 cells treated withA.gigantifoliaStapf active constituent for 24 and 72 h (4, 8 μg/mL) increased, especially at 72 h, while the phosphorylated protein levels of JNK, ERK and p38 decreased significantly. It is suggested that the anti-hepatoma effect of the active components ofA.gigantifoliaStapf may be related to the increase of the expression of DUSP1, DUSP4 and DUSP5 in cells and the decrease of the phosphorylation level of ERK, JNK and p38. It can significantly inhibit the proliferation of HepG2 cells, induce apoptosis of hepatoma cells, inhibit invasion and metastasis, and has a strong anti-hepatoma effect. The water extract ofA.gigantifoliaStapf showed obvious inhibitory effect on HepG2 and SK-hep1 cells, and theIC50was (3.21 ±0.35) and (2.17 ±0.10) mg/mL, respectively. Under the inverted fluorescence microscope, it was found that the water extract ofA.gigantifoliaStapf could significantly induce the apoptosis of two kinds of hepatocellular carcinoma cells. It can decrease the expression of CDK2 mRNA and increase the expression of Caspase-3 and E-cadherin mRNA in HepG2 cells, suggesting that the water extract ofA.gigantifoliaStapf may inhibit the occurrence and development of hepatocellular carcinoma by down-regulating the expression of CDK2 and up-regulating the expression of Caspase-3 and E-cadherin.

Yao Zhirenetal.[26]found that ethyl acetate fromA.gigantifoliaStapf could significantly inhibit the proliferation of human hepatoma cell line HepG2, down-regulate the expression of Bcl-2, up-regulate the expression of Bax, down-regulate the expression of Caspase-3, Caspase-8 and Caspase-9, and up-regulate the expression of cleaved-Capase-3 and cleaved-Capase-9. It shows significant anti-hepatoma effect, and its mechanism is to up-regulate Bax and down-regulate Bcl-2, activate cleaved-Capase-3 and cleaved-Capase-9, and induce apoptosis of HepG2 cells.

Mu Lihuaetal.[27]studied the antitumor activity and potential molecular mechanism of triterpenoid saponin AG36 isolated fromA.gigantifoliaStapf against human breast cancer cells MCF-7, MDA-MB-231 and SK-BR-3. It was found that AG36 inhibited the viability of MCF-7, MDA-MB-231 and SK-BR-3 cells in a dose-and time-dependent manner, and theIC50at 48 h was about 0.73, 18.1 and 23.4 μM, respectively. AG36 significantly induced apoptosis and G2/M phase arrest in three kinds of breast cancer cells. In addition, AG36 decreased the expression of cycle regulatory proteins cyclin B1 or cyclin D1. In MCF-7 and MDA-MB-231 cells, AG36 strongly increased the expression of cleaved caspase-3 and-8 protein, while in SK-BR-3 cells, AG36 only increased the expression of cleaved caspase-3 protein. The Bax/Bcl-2 ratio and cytochrome c content in three kinds of breast cancer cells were significantly higher than those in the control group. Westernblot detection of death receptor related proteins Fas/FasL, TNFR1 and DR5 showed that different breast cancer cells activated the exogenous caspase-8 pathway mediated by death receptor through different receptors. In addition, caspase-8 inhibitor z-IETD-fmk could significantly block AG36-induced apoptosis in MCF-7 cells.Invivostudies showed that compared with the control group, AG36 significantly inhibited the growth of MCF-7 xenografts in BALB/c nude mice. In short, AG36 inhibited the proliferation of MCF-7, MDA-MB-231 and SK-BR-3 cells through endogenous mitochondria and exogenous death receptor pathways. AG36 may be a potential therapeutic agent for breast cancer.

Mu Lihuaetal.[28]also found that AG36 showed significant cytotoxicity to breast cancer cells bothinvitroandinvivo, and further evaluated the anti-angiogenic effect of AG36 and its potential mechanism. The results showed that AG36 could significantly inhibit the proliferation, migration and invasion of human umbilical vein endothelial cells (HUVEC). Further studies on the molecular mechanism of anti-angiogenesis showed that AG36 significantly inhibited the phosphorylation of FAK and AKT in HUVECs, and down-regulated the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2). Pretreatment with PI3K inhibitor (LY294002) and FAK inhibitor (PF562271) could significantly enhance the inhibition of HUVEC proliferation and p-FAK by AG36, respectively. In addition, AG36 inhibited tumor growth in xenotransplantation model and inhibited the expression of p-VEGFR2 and p-Aktinvivo.Molecular docking simulations show that AG36 forms hydrogen bonds and hydrophobic interactions in the ATP binding domain of VEGFR2 kinase domain. This suggests that AG36 may be a potential anti-angiogenic cancer therapy or a major drug candidate.

Mu Lihuaetal.[29]found that triterpenoid saponin AG8 isolated fromA.gigantifoliaStapf could induce apoptosis of triple negative breast cancer cells through oxidative stress, which inhibited the proliferation of human breast cancer MDA-MB-231 cells. Further, the role of AG8 in different types of triple negative breast cancer (TNBC) cells was analyzed, including MDA-MB-231, BT-549 and MDA-MB-157 cells. AG8 inhibited the viability of MDA-MB-231, BT-549 and MDA-MB-157 cells in a dose-dependent manner, and its cytotoxicity to African-American (AA) and mesenchymal (M) subtypes was stronger than that of Caucasian (CA) and mesenchymal stem cell-like (MSL) subtypes. AG8 impairs CMXRos uptake by mitochondria of TNBC cells in a dose-dependent manner, which can be recovered by N-acetyl-1-cysteine (NAC). AG8 affects the levels of GSH, SOD and MDA in TNBC cells, but different TNBC subtypes have different sensitivities to AG8 and NAC. In addition, it was found that AG8 increased the Bax/Bcl-2 ratio and cytoplasmic cytochrome c level of BT549 and MDA-MB-157 cells, and significantly decreased the phosphorylation of ERK and AKT. AG8 stimulates its anticancer effect through ROS production, ERK and AKT activation, and triggering mitochondrial apoptosis pathway in TNBC cells. AG8 has selective cytotoxicity on AA and M-TNBC subtypes, and significantly induces apoptosis of MDA-MB-157 (AA subtype) cells through a pathway independent of ROS, which is different from the other two subtypes. Its potential mechanism needs to be further studied.

Ping Wenetal.[30]isolated four new oleanane triterpenoid saponins from theA.gigantifoliaStapf. Among them, ardisiacrispin A，cyclamiretinA 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside，cyclamiretin A 3β-O-α-L-rhamnopyranosyl-(1→3)-[β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→2)]- β-D-glucopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranoside showed good antitumor activity against NCI-H46, SF-268, MCF-7 and HepG2 cells.

2.5 Antitussive and expectorant effectHuang Yongyietal.[31]found that alcohol extract ofA.gigantifoliaStapf could significantly increase the latency of cough, reduce the frequency of coughing in 2 min, increase the excretion of phenol red in the trachea of mice, and show obvious antitussive and expectorant effects.

3 Discussion

In recent years, the research onA.gigantifoliaStapf has been gradually deepened and some progress has been made. In the study of chemical composition, more than 50 components such as phenols, quinones, coumarins, and triterpenes have been isolated fromA.gigantifoliaStapf, and there are more than 60 kinds of volatile oil. The results showed that the content of polysaccharides, total saponins, alkaloids and quercetin inA.gigantifoliaStapf was about 2.30%, 1.01%, 0.28% and 1.38%, respectively.

As the saying goes, "Two feet cannot be separated fromA.gigantifoliaStapf ", it can be seen that it plays a good role in relieving fatigue, promoting blood circulation, and so on[1]. It is also widely used in clinical injury, fracture, hyperosteogeny, rheumatoid arthritis and so on. Most of the components isolated fromA.gigantifoliaStapf have pharmacological activity, for example, phenols and coumarins have anti-inflammatory and antioxidant effects, polysaccharides have blood-activating and anti-thrombotic effects, triterpenoid saponins have anti-tumor effects, and alcohol extracts can also resist rheumatoid arthritis. These pharmacological effects are verified by the application ofA.gigantifoliaStapf in injuries from falls, promoting blood circulation and relieving pain and rheumatoid arthritis. However, more than 10 triterpenoids isolated fromA.gigantifoliaStapf showed good inhibitory effect on nearly 10 kinds of tumor cells and had good anti-tumor activity, especially compounds AG4, AG3, AG8 and AG36. Biotransformation method has been used to improve the yield of these components[17], which shows thatA.gigantifoliaStapf has a good prospect of development and application in anti-tumor field. Therefore, we should maintain the sustainable development and research, analyze the mechanism of specific effective components, synthesis mechanism and action mechanism, and explore the new pharmacological effects relating toA.gigantifoliaStapf, so as to provide a theoretical basis for the full development and utilization ofA.gigantifoliaStapf resources.