Biological activities and stability testing of a Thai traditional remedy for female reproductive cancer treatment

Ya Kae Ma Reng Nai Mod Look (MRM) is Thai traditional medicine used to treat female reproductive cancer described in the Thai traditional medicine book by Krom Luang Chumphon Khet Udomsak. It consists of Smilax corbularia Kunth., Dioscorea membranacea Pierre ex Prain & Burkill., Stemona collinsiae Craib., Rauwenhoffia siamense Scheff., Canna indica L., and sodium chloride. It has not ever been researched into the biological activity of this remedy. Therefore, this study aimed to investigate the cytotoxic, antioxidant, anti-inflammatory, and antifungal activities of MRM extracts and its plant ingredients. Furthermore, this study determined the marker compound and also tested the stability of MRM extract.The ethanolic extracts of MRM and its plant ingredients were extracted by maceration with 95% ethanol, and decoction which gave the ethanolic and aqueous extracts of MRM and its plant ingredient extracts. The water extract of C. indica (CIW) had the highest yield of 49.37% w/w. The yields of ethanolic extracts of MRM (MRME) and water extract of MRM (MRMW) were 11.09 and 47.70% w/w, respectively.All extracts were investigated cytotoxic activity against three cancer cell lines including endometrioid adenocarcinoma cell line (HEC-1-A), cervical cancer cell line (HeLa), ovarian cancer cell line (SKOV-3), and two normal cell lines, including normal lung fibroblast line (MRC-5) and human keratinocyte cell line (HaCaT). The result found that MRME and MRMW had no cytotoxic effect against HEC-1-A and SKOV-3. Nevertheless, MRME was highly toxic to HeLa cells with an IC50 value of 0.1 µg/mL, while MRMW had no toxicity to this cell line. Meanwhile, the plant ingredients of MRME, including the ethanolic extract of Dioscorea membranacea (DME), the ethanolic extract of Stemona collinsiae (SCLE), and the ethanolic extract of Rauwenhoffia siamense (RSE) showed cytotoxicity against cancer cell lines. The results found that RSE, DME, and SCLE exhibited significantly inhibitory effect on the growth of HEC-1-A with IC50 values of 30.06±0.79, 48.26±4.88, and 69.29±4.47 µg/mL, respectively. In addition, the RSE exhibited the highest cytotoxic activity against HeLa with IC50 of 0.01µg/mL, followed by SCLE, DME, and the water extracts of Rauwenhoffia siamense (RSW) showed cytotoxic effect with IC50 of 12.59±0.88, 35.80±1.45, and 84.48±2.24 µg/mL, respectively. Moreover, the RSE also showed significant cytotoxicity against SKOV-3 with IC50 of 54.46±0.67 µg/mL. Besides, RSE showed the highest toxicity significantly to MRC-5 with IC50 of 0.004±0.00 µg/mL, followed by MRME, SCLE, DME, and RSW with IC50 of 0.10±0.02, 36.84±4.07, 71.06±0.13, and 79.37±1.75 µg/mL, respectively. Meanwhile, the results of cytotoxic activity against HaCaT showed that only RSE and DME was moderate cytotoxic activity against HaCaT with IC50 of 24.12±0.64 and 90.57±0.13 µg/mL. For antioxidant activities of MRM and its plant ingredients extracts found that MRME was higher ferric-reducing power than MRMW, with FRAP and TEAC values of 384.15±9.18 mgFe2+/g extract and 144.59±3.54 mgTrolox/g, respectively. The RSW showed the highest FRAP and TEAC value with 881.40±26.03 mgFe2+/g extracts and 336.19±10.03 mgTrolox/g, followed by SCE, RSE, CIE, DME, and SCLE, respectively. Moreover, astilbin, the chemical marker of MRM showed the highest FRAP value and TEAC value of 1,227.82±17.51 mgFe2+/g extract and 572.98±9.25 mgTrolox/g, respectively. Furthermore, MRME had good superoxide anion scavenging activity with IC50 of 45.06±7.80 µg/mL, while MRMW had no effect. Interestingly, the RSE had the highest significant superoxide radical scavenging activity with IC50 of 21.30±1.34 µg/mL, followed by RSW, SCE, CIE, SCW, and CIW, respectively.MRME had a better inhibition effect on nitric oxide production than MRMW, with IC50 of 45.93±2.70 and more than 100 µg/mL, respectively. Moreover, the DME had the highest significant effect on inhibiting nitric oxide production with IC50 of 15.75±2.45 µg/mL, followed by RSE, SCLE, and CIE. In addition, this study investigated for inhibition of IL-6 production in RAW 264.7 cells using sandwich ELISA and found that MRME had the highest inhibition effect of IL-6 cytokine with IC50 of 35.47±3.29 µg/mL. Still, MRMW showed no inhibition effect on IL-6. On the other hand, DME and RSE inhibited IL-6 production with IC50 values of 39.57±2.34 and 99.39±0.09 µg/mL, respectively. Furthermore, MRME, MRMW, and astilbin had no inhibition effect on TNF- α releasing with IC50 more than 100 µg/mL.The antifungal activity results found that MRME inhibited C. albicans with a MIC value of 2.5 mg/mL and MFC value of more than 5 mg/mL. Moreover, RSE had good antifungal activity against C. albicans with a MIC value of 0.625 mg/mL and DME inhibited C. albicans with a MIC value of 2.5 mg/mL and MFC value of 5 mg/mL.The results of the biological activity of MRME showed high antioxidant activity. The main compound in the MRME was astilbin, a flavonoid by astilbin contents in MRME extracts was 32.54 ± 0.90 mg/g of extract. MRME was investigated through stability testing under the accelerated condition at 40±2°C with 75±5% RH for six months. After that, all samples were determined for cytotoxic activity against cervical cancer cells (HeLa) by SRB assay, antioxidant activity by FRAP assay, and anti-inflammatory activity by inhibiting nitric oxide production assay. The results of MRME showed a significantly decreased cytotoxic activity against Hela cells after 60 days. However, the cytotoxic activity of MRME under accelerated had not significantly difference after 180 days. The antioxidant activity of MRME under accelerated conditions showed that FRAP values and TEAC values of MRME were less changed after 180 days. Moreover, the inhibition of nitric oxide production of MRME was investigated. The results showed that the inhibition effect of MRME on nitric oxide production decreased significantly after 30 days. It showed no inhibition effect on nitric oxide production with an IC50 value was more than 100 µg/mL.These results can be concluded that MRME showed the greatest inhibition specific to cervical cancer cells. Its plant ingredients, including SCLE and DME, were active cytotoxicity against cervical cancer cell lines. Furthermore, MRM remedy extract and its plant ingredients showed good antioxidant, anti-inflammation, and antifungal activities, which would be related to the effect of anti-cancer. The primary compound of MRME was astilbin which is used to determine the marker of quality control for antioxidant activity. Thus, these results can support preliminary medicinal properties of this remedy for cancer treatment, but relevant MRM biological activities and safety in animal model should be further studied.