Antiproliferative activities and phenolic acid content of water and ethanolic extracts of the powdered formula of Houttuynia cordata Thunb. fermented broth and Phyllanthus emblica Linn. fruit

Nowadays, cancer is the leading cause of death in many countries of the world. Chemotherapy is a common treatment strategy for advanced and metastatic cancer. However, the cost of chemotherapy is a major concern in developing countries. In addition, resistance and negative side effects resulting from chemotherapy with conventional anti-cancer drugs pose some limitations in clinical use. Consequently, herbal medicine remains an inevitable alternate or adjunct therapy for cancer prevention and treatment. Various plant components with bioactive compounds and derived natural products are being intensively investigated to explore their potential for prevention and treatment of cancer. Houttuynia cordata Thunb. (known in Thai as Plu-Kao) is a medicinal herb indigenous to many parts of East and Southeast Asia including Thailand [1, 2]. The plant has been recognized as possessing many biological and pharmacological properties including anticancer, antioxidative, antiviral and immunomodulatory activities [3]. The fermented H. cordata has been demonstrated to exhibit cytotoxic effects on human leukemia cells and hepatocellular cancer cells to a considerable extent [4, 5].

Phyllanthus emblica Linn. or Emblica officinalis Gaertn. (known in Thai as Ma-kham-pom) is another medicinal plant used in Asian traditional medicine systems against different ailments including peptic ulcer, inflammation, dyspepsia and cancer, etc. [6, 7]. Its fruits are traditionally consumed for its immense nutritive values as well. Experimental evidence suggested that its fruit had several phytochemicals such as gallic acid, ellagic acid and pyrogallol that possess antineoplastic effects [6]. It has also been reported to possess immunomodulatory, chemomodulatory, radiomodulatory, antioxidative, anti-inflammatory and antimutagenic activities, which directly or indirectly prevent carcinogenesis [6, 7]. A number of in vitro and in vivo experiments point out that fruit extract of P. emblica has potent anticarcinogenic property [8‐11]. A recent study has indicated that P. emblica extract inhibits ovarian cancer cell proliferation both in vitro and in vivo through inhibiting angiogenesis and inducing autophagy [12]. Furthermore, the bioactive compound gallic acid, present in fruit of P. emblica causes cell death through induction of apoptosis [13]. The bioactivities of P. emblica extract are mainly mediated by polyphenols [10]. Many phenolics have been documented to have potent antioxidant, anticancer, antibacterial, antiviral, and anti-inflammatory potentials [14]. The fruit of P. emblica, being rich in natural antioxidants with free radical scavenging potential, might prevent reactive oxygen species from DNA damage and carcinogenesis [10, 11, 15]. The anti-inflammatory activity of P. emblica extracts might deter inflammation-related cancer [10]. It has also been mentioned that ethanolic extract of P. emblica fruit showed antiproliferative and antioxidant activities, whereas methanol extract showed chemopreventive potential against hepatocarcinogenesis [16‐18].

In recent times, the H. cordata fermented broth has been widely used in Thailand as a dietary supplement, however, the consumption may be limited due to its unpleasant taste. Therefore, a powdered formula of H. cordata fermented broth and P. emblica fruit has been developed. This is very interesting from a health perspective and thus, anticancer effects and chemical composition of this powdered formula needs to be investigated to provide scientific information to the public. In the present study, we evaluated antiproliferative activity of the powdered formula extracts in a panel of cancer cell lines and identified several phenolic compounds from ethanolic and water extracts of the powdered formula with HPLC and LC-MS techniques.

A large number of cancer patients in developing countries face problems with the high cost of chemotherapy which induces researchers to develop herbal medicine as an alternative option. Poor patients exploit traditional medicinal plants containing unknown bioactive constituents to protect their lives. Moreover, phenolic compounds present in vegetables and fruits are reported to have chemopreventive effects. Findings from previous studies suggested that the anticancer activity of the H. cordata fermentation products was partly attributed to the presence of p-coumaric, ferulic and sinapinic acids [20]. The phenolic components of P. emblica have been associated with inhibition of MCF7 cells in vitro [21]. Phenolic acids in P. emblica fruits showed strong antiproliferative activity against MK1 and HeLa cells [22]. Jose et al. reported that aqueous extract of P. emblica fruit showed cytotoxicity against L929 cells in a dose-dependent manner and reduced tumor volume of the solid tumors [9]. They also reported that antitumor activity of the extract may be partially related to its ability to regulate cell cycle progression. Accordingly, one may envision the powdered formula of H. cordata fermented broth and P. emblica fruit as a combination formula with promising anticancer activity. To our knowledge, we are the first who demonstrated that the powdered formula of H. cordata fermented broth and P. emblica fruit had effects on cell growth, apoptosis and cell cycle arrest. Most interestingly, both ethanolic and water extracts of the powdered formula were more potent against all cancer cell lines tested than the lyophilized water-soluble constituents of H. cordata fermented broth alone [20]. This observation may be related to the greater amounts of phenolic compounds especially p-coumaric and ferulic acids in the extracts (Table 1) compared with that in the lyophilized water-soluble constituents of H. cordata fermented broth alone [20].

Our results indicated that the water and ethanolic extracts reduced viability of HeLa, HT29 and MCF7 cells mainly by triggering apoptosis. Though all the cancer cells underwent apoptosis due to treatment with the extracts, antiproliferative effects of HCT116 and Jurkat cells were linked with cell cycle arrest at S and G0/G1 phases, respectively. In line with our findings, lyophilized water-soluble constituents of fermentation products from H. cordata suppressed the growth of HeLa, HCT116, and HT29 cells in a concentration- and time-dependent manner by inducing apoptosis [20]. Previous studies showed that P. emblica could induce apoptosis in mouse and human carcinoma cell lines including Dalton’s lymphoma ascites and CeHa cell lines, HeLa cells, SK-OV3 and HepG2, A549, SW620 [11, 23]. Growth inhibitory activity of P. emblica fruit extract was primarily demonstrated through induction of apoptotic cell death. P. emblica fruit extract induced apoptosis in HeLa cells by a death receptor-mediated mechanism and invasiveness of MDA-MB-231 cells in vitro. In addition, application of P. emblica extract on mouse skin resulted in reduction of tumor numbers and volumes [11]. Its anticancer activity was also related with inhibition of activator protein-1 (AP-1) which targeted transcription of viral oncogenes responsible for cervical cancer [24]. It has been reported that polyphenols enriched P. emblica berry extract or simple aqueous extracts have shown cytotoxic activity against ovarian cancer cells [25]. Zhu et al. mentioned that the extract of P. emblica containing polyphenols was capable of inhibiting cell proliferation in HeLa cells, accompanied by cell cycle arrest at G2/M phase and apoptotic cell death [26].

Moreover, in the present study, several phenolic acids were identified in the powdered formula extracts and their concentration was determined. The identified phenolic acids of both water and ethanolic extracts of the powdered formula were gallic, p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids where the amount of p-coumaric and ferulic acids were greater than other identified compounds (Table 1). Similar to our study, a number of phenolic acids including protocatechuic, p-hydroxybenzoic, and vanillic, syringic, p-coumaric, ferulic, and sinapinic acids were identified in whole plant methanolic extract and water-soluble constituents of H. cordata fermentation products [20, 27]. Water extract of P. emblica was found to contain polyphenol contents [11, 28]. Many studies showed that phenolic acids were able to inhibit cancer cell growth by induction of apoptosis and/or cell cycle arrest. Gallic acid has been reported to inhibit the growth of prostate cancer cell lines (IC₅₀ values = 15.6–20.7 μg/mL for 48 h exposure) [29]. p-Hydroxybenzoic acid has not yet been shown to suppress the growth of any cancer cell line, however, its derivatives exhibited anticancer activity [30]. p-Coumaric acid inhibited the growth of the colon cancer cell lines (HCT-15 and HT29 cells; IC₅₀ values = 1400 and 1600 μM, respectively, for 48 h exposure) by triggering apoptosis in an ROS-dependent mitochondrial pathway [31]. Vanillic acid has not yet been shown to suppress the growth of any cancer cell line, however, vanillin has been shown to induce both apoptosis and cell cycle arrest in HT29 cells [32]. Syringic acid has been found to have anticancer potential against T47D breast cancer cells [33]. Sinapinic acid inhibited the growth of several human cancer cells (HT29, HCT116, HeLa and Jurkat cells; IC₅₀ values = 1.6, 2.1, 0.9 and 2.8 mM, respectively, for 72 h exposure) [19]. Ferulic acid has been shown to inhibit the growth of several human cancer cells (HT29, HCT116, MCF7 and HeLa cells; IC₅₀ values = 2.2, 2.1, 4.0 and 0.9 mM, respectively, for 72 h exposure) by apoptosis induction [34, 35]. Accordingly, gallic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids may, at least in part, contribute to antiproliferative activity of the extracts from the powder mix of H. cordata fermented broth and P. emblica fruit especially those with relatively high amounts in the extracts (p-coumaric and ferulic acids). Our findings on Vero cells were in agreement with the report by Ngamkitidechakul et al. who demonstrated P. emblica extracts were non-toxic against normal lung fibroblast MRC5 cells [11]. Further investigation on the use of these extracts in combination with other chemotherapeutics and natural products may help to develop more effective therapeutic approaches.

The present study demonstrated that ethanolic and water extracts from the powdered formula of H. cordata fermented broth and P. emblica fruit were able to prevent cancer cell growth through apoptosis and cell cycle arrest. This powdered formula is a good source of phenolic compounds especially p-coumaric and ferulic acids. Phenolic acids were perhaps responsible for growth inhibition of cancer cells. From the findings in this study, further research should be conducted on the use of powdered formula of H. cordata fermented broth and P. emblica fruit as a complementary medicine for cancer treatment and prevention in both animal models and clinical trials.