DOI: 10.14457/tu.the.2016.1482
title: In vitro cytotoxic activity against human lung cells of A-Phai-Sa-Lee remedy and its ingredient

description: A-Phai-Sa-Lee remedy [APSL] is Thai traditional medicine for longevity or rejuvenation and claimed to improve blood circulation. It is listed in NLEM [National List of Essential Medicine, 2013] that recommended to treat indigestion, flatulence and distention. This remedy composed with 19 plant species. It has been used to treat symptoms of COPD and  lung care as an inflammatory in lung cancer. The objective of this study is to investigate cytotoxic activity of [APSL] extracts and its ingredient herbs against three different type of cancer cell such as human lung adenocarcinoma cell line [A549], human lung squamous carcinoma cell line [NCI-H226] and human large cell lung carcinoma cell line [COR-L23] and one normal lung cell. APSL and its ingredient plants were extracted by maceration in 95% ethanol and boiling in water. All extracts were tested for cytotoxic activity by sulforhodamine B [SRB] assay. Results of these show that the ethanolic extract of  [APSL] exhibited cytotoxic activity against three human lung cancer A549, NCI-H226 and COR-L23 and comparation with lung normal cell (MRC5) by selective index (SI) ( IC50 values of 34.75±1.93[SI=2.44], 88.72±2.93 [SI=0.95]and 10.16±1.67 [SI=8.34]μg/ml, respectively. The results of A549 and NCI-H226 were classified as less-active according to the NCI guided line but the result of COR-L23 was classified as active. In ethanolic extract of Clausena excavate showed the highest cytotoxic activity against A549, NCI-H226 and COR-L23 with IC50 values of 1.14±0.30 [SI=0.70], 10.46±3.93 [SI=0.08], 1.18±0.06 [SI=0.68] μg/ml, respectively. Following the ethanolic extract of Plumbago indica showed against A549, NCI-H226 and COR-L23 with IC50 values of8.38±0.24 [SI=1.93], 11.36±1.80 [SI=1.42], and 6.18±0.49 [SI=2.61] μg/ml, respectively. However, the water extract of APSL had  no cytotoxic against three cancer cells. The water extracts of Clausena excavate showed the highest  cytotoxic activity against A549 and COR-L23 with IC50 values of 28.12±2.25 [SI=0.95] and 26.99±0.14 [SI=0.99] μg/ml, respectively. These results support the use of A-Phai-Sa-Lee remedy in Thai traditional medicine for preventing of lung cancer. Clausena excavate and Plumbago indica are recommended as active ingredient of this remedy.  Its  extracts have the potential for developing as new anti-cancer drugs for human health.Chemical analysis in APSL by HPLC showed that piperine is a main compound in the ethanolic extract of APSL because it showed the highest content [104.64±7.77 mg/g of extract].  The second is eugenol [98.83±6.09 mg/g of extract].  From this result can be used for supporting quality control from chemical fingerprint by using RP-HPLC method.The 95% ethanolic extract of C. excavata which the highest cytotoxic activity was selected for isolation of the active cytotoxic activity compound by bioassay guided fractionation. Sitostenone, β-sitosterol and stigmasterol were isolated and showed less-active results of cytotoxic activity with three cancer cells. The IC50 of Sitostenone against A549, NCI-H226 and COR-L23 were 41.11±1.57, 64.87±3.51 and 24.17±3.86 μg/ml, respectively. The IC50 of combination of β-sitosterol and stigmasterol with A549, NCI-H226 and COR-L23 were 58.94±1.40, 61.41±5.87 and 25.94±3.51 μg/ml, respectively.Stability of cytotoxic activity of the 95% ethanolic extracts of APSL remedy showed high activity agianst human lung large cell line [COR-L23] with IC50 values in range of 10.16-32.02 µg/ml.  For human lung adenocacinoma cell line [A549] showed IC50 values in range of 25.07-45.14 µg/ml These results indicated that the ethanolic extract of APSL under accelerated condition at 40±2oC with 75±5% RH for 6 months [Days 15, 30, 60, 90, 120, 150 and 180] were stable because IC50 of day0 and day180 showed no significant different [p-value &gt;0.05]. However, the stability of cytotoxic activity of APSL remedy against human lung squamous carcinoma cell line [NCI-H226] showed IC50 values in range of 35.68-88.72 µg/ml that unstable because IC50 of day0 and day180 showed significant different [p-value &lt;0.05].In conclusion, the 95% ethanolic and water extract of APSL remedy revealed good cytotoxicity for COR-L23. Therefore, these results could support that APSL used for lung cancer treatment. However, this remedy should be continuously developed as cancer product for lung cancer cancer treatment