DOI: 10.14457/tu.the.2015.615
title: Immunomodulatory effect of Thai medicinal preparation for treatment of cancer patients

description: Benjakul and Hua-Khao-Yen are Thai medicinal preparations which have long been used for cancer treatment. Benjakul is used as adaptogenic drug before using other cancer drugs. This preparation consists of five plants; the fruits of Piper chaba Hunt., the roots of Piper sarmentosum Roxb., the stems of Piper interruptum Opiz, the roots of Plumbago indica Linn. and the rhizomes Zingiber officinale Rosc. Benjakul preparation and its plant components have been reported for cytotoxic activity on cancer cell lines but they have not been reported on immunomodulatory activity. Five species of Hua-Khao-Yen have been found five species in Thailand, but two species of Hua-Khao-Yen are popularly used as drugs for cancer patients, namely Smilax corbularia Kunth and Dioscorea membranacea Pierre. Two species of Hua-Khao-Yen have been reported regarding some immunomodulatory pathways. Thus, the objectives of this study were to investigate the immunomodulatory activity of Benjakul, two types of Hua-Khao-Yen and their isolated compounds. The finding results may be used for the basic data of Benjakul preparation and Hua-Khao-Yen for immunomodulatory aspects of cancer treatment.Benjakul and Hua-Khao-Yen were extracted by two methods. Decoction method in water to obtained as water extract and maceration in 95% ethanol to obtain on ethanolic extract. Bioassay guided fractionation was used to guide isolation of active compounds. The immunomodulatory assay of extracts and compounds were performed to study the effect of NK cells activity and lymphocyte proliferation. The stimulatory effect on cytokine productions, IL-2 and IFN-γ, were investigated by ELISA technique. The inflammatory and anti-inflammatory cytokines activation by LPS-stimulated RAW264.7 cells also were detected by ELISA. Finally, safety studies of component plants in Benjakul preparation were investigated by in vitro and in vivo experiments. Piper species were investigated for their effect on TRPV1 and TRPA1 activation, adjuvant effects, the stimulation of trafficking dendritic cells and cytokine production in FITC sensitized mice.The ethanolic extract of S. corbularia showed significant effects on NK cells activity and lymphocyte proliferation at low concentration (0.01 and 0.1 μg/ml) and it decreased NK cells activity and lymphocyte proliferation at high concentration. Moreover, the ethanolic extract of S. corbularia significantly increased IFN-γ production (1-10 μg/ml). The water extract did not significantly affect stimulation of NK cells, lymphocyte proliferation and IL-2 and IFN-γ production. For cytokine production from macrophage, the ethanolic and water extracts of S. corbularia had no effect on inflammatory and anti-inflammatory cytokines. Two isolated compounds from the ethanolic extract of S. corbularia, quercetin and astilbin, had no significant effect on NK cells activity. Quercetin also showed no significant stimulation on lymphocyte proliferation. The ethanolic extract and water extract of D. membranacea were investigated for their immunomodulatory activity. The results showed that the ethanolic extract of D. membranacea significantly increased NK cells activity at low concentration (0.1 μg/ml). Moreover, the ethanolic extract of D. membranacea showed inhibitory effect on IL-1β and IL-6 production (IC50 =  46.82 and 0.90 μg/ml) and activated IL-10 production (concentration in range 10-50 μg/ml to be 1.10-1.34 times when compare with condition in the presence of LPS) by LPS-stimulated RAW264.7 cells. Futhermore, the water and ethanolic extracts of D. membranacea had significant effect on IL-2 and IFN-γ production by PHA-stimulated PBMCs. Two compounds namely, dioscorealide B and 2,4 dimethoxy-5,6 dihydroxy-9,10 dihydrophenanthrene were isolated from the ethanolic extract of D. membranacea. These compounds showed no significant stimulation on NK cells and lymphocyte proliferation. 2,4 dimethoxy-5,6 dihydroxy-9,10 dihydrophenanthrene also showed inhibitory effect on IL-1β and IL-6 production with IC50 value of 4.38 and 9.30 μg/ml. Dioscorealide B showed inhibitory effect on IL-6 production with IC50 value of 8.59 μg/ml. However, dioscorealide B and 2,4 dimethoxy-5,6 dihydroxy-9,10 dihydrophenanthrene had no stimulatory effect on IL-10 production from LPS-stimulated RAW264.7 cells.The ethanolic extract of Benjakul caused an increase of NK cells activity, lymphocyte proliferation, IL-2 and IFN-γ production at low concentrations (concentration in range 0.01-10 μg/ml). It also showed inhibitory effect on IL-1β and IL-6 with IC50 value of 62.93 and 51.83 μg/ml, respectively. Piperine which is an isolated compound from the ethanolic extract of Benjakul, showed no significant effect on NK cells activity. However, piperine inhibited IL-6 with IC50 value of 16.71 μg/ml. From these results, the ethanolic extract of Benjakul showed anti-inflammatory effect but it had less effect on NK cells and lymphocyte proliferation. The ethanolic extract of P. chaba, P. interruptum and P. sarmentosum and piperine were chosen to safety study. Three Piper species are component plants of Benjakul preparation. The results showed that piperine and the ethanolic extracts of P. chaba and P. interruptum activated TRPV1 and TRPA1 receptor which affect to burning pain and stomachache. Furthermore, piperine and P. chaba extract showed adjuvant effect and induced contact hypersensitivity in mouse ear swelling test. The ethanolic extract of P. chaba enhanced dendritic cells-trafficking to draining lymph nodes cells and induced IL-4 and IFN-γ production in FITC-sensitized mice. Even though piperine induced adjuvant effect, it could not activated dendritic cells-trafficking and cytokines production (IL-4 and IFN-γ) in FITC-sensitized mice.These results can be summarized in that Hua-Khao-Yen-Tai or D. membranacea showed the strongest effect on immune system when compared with Hua-Khao-Yen-Nua or Smilax corbularia and Benjakul preparation. Hua-Khao-Yen-Tai showed stimulatory effect on NK cells and lymphocyte proliferation. Moreover, Hua-Khao-Yen-Tai also inhibited inflammatory cytokine (IL-1β and IL-6) and activated anti-inflammatory cytokine (IL-10). However, Hua-Khao-Yen-Nua also showed some pathway of immunomodulatory activity by activation on NK cells, lymphocyte proliferation and IFN-γ production. For Benjakul preparation, it showed no effect on NK cells activity and lymphocyte proliferation. However, Benjakul showed anti-inflammatory activity which inhibited IL-1β and IL-6. Thus, Benjakul can be used as an anti-inflammatory drug for cancer patients. However, the side effect of Benjakul should be concern because the component plant in Benjakul preparation, P. chaba, activated TRP channel and contact hypersensitivity. Thus, Benjakul preparation may show side effect similar to P. chaba extract. However, the ingredients of all extracts exhibited less immunological activity than their crude extract. Thus, the ethanolic extract of Hua-Khao-Yen and Benjakul should be promoted for their immunomodulatory and anti-inflammatory effects on cancer treatment.