DOI: 10.14457/tu.the.2015.650
title: Biological activities of Thai traditional remedy called Kheaw-Hom and its plant ingredients

description: Kheaw-Hom is a Thai traditional remedy which folk doctors have long used to treat fever in the exanthematous fever group such as chickenpox, measles, herpes zoster and hand, foot and mouth disease. It consists of eighteen Thai medicinal plants as follows: Phim sen ton (Pogostemon cablin (Blanco) Benth.), Phak krachom (Limnophila rugosa Merr.), Mak phu (Cordyline fruticosa (L.) Goeppert.), Mak mia (Cordyline fruticosa (L.) Goeppert.), San phra hom (Eupatorium stoechadosmum Hance), Faek hom (Vetiveria zizanioides (L.) Nash ex Small), Proh hom (Kaempferia galanga Linn.), Chan thet (Myristica fragrans Houtt.), Chan dang (Dracaena loureiri Gagnep.), Wan kep rat (Angiopteris evecta (G.Forst) Hoffm.), Wan ron thong (Globba malaccensis Ridl. ), Nae ra phu sri (Tacca chantrieri Andre), Phit sa nat (Sophora exigua Craib), Ma has sa dam (Cyathea gigantea Holtt.), Phi kul (Mimusops elengi Linn.), Bun nak (Mesua ferrea Linn.), Sa ra phi (Mammea siamensis  Kosterm. ) and Bua luang  (Nelumbo nucifera Gaertn.). Some plant ingredients in this remedy have been investigated for antimicrobial and anti-inflammatory activities. However, for Kheaw-Hom remedy, there is no report to verify this. Consequently, the objectives of this research were to study the antiviral, antimicrobial and anti-inflammatory activities that are related to exanthematous fever and skin infection complications of ethanolic and aqueous extracts of Kheaw-Hom remedy and its plant ingredients. All extracts were tested for antiviral activity by antiviral activity based CPE assay, antimicrobial activities by using disc diffusion method and microtitre plate-based antimicrobial assay and anti-inflammatory activities by determination of inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cell lines. The extract which showed the strongest activity was selected to study for stability testing.   Kheaw-Hom remedy and each of its plant ingredients were extracted by maceration in 95% ethanol and decoction in water to obtain ethanolic and aqueous extracts, respectively. The percentage yields of the ethanolic and aqueous extracts of Kheaw-Hom remedy were 8.75% and 13.36%, respectively. The highest percentage yield of the ethanolic extract was D. loureiri (19.49%) and the highest percentage yield of the aqueous extract was M. siamensis (30.56%).  All extracts were tested for biological activities. First, antiviral activity was tested by using the antiviral activity based cytopathic effect (CPE) assay of enterovirus 71 (EV71) on Vero cell lines. The aqueous extracts of Kheaw-Hom remedy and its plant ingredients which cytotoxicity was less than 20%, were tested for antiviral activities against 100, 50 and 25 TCID50 (50% tissue culture infective dose) EV71 in duplicate experiments. Morphological changes of Vero cells which were infected with EV71 at 100, 50 and 25 TCID50 were observed after incubated 5 days. The results showed that the aqueous extract of Kheaw-Hom remedy (KHA) had antiviral activity against 25 TCID50 EV71 with cytopathic effect less than 50% at a concentration 400 μg/ml. In contrast, the ethanolic extract of Kheaw-Hom remedy (KHE) exhibited toxic to Vero cells. The aqueous extract of T. chantrieri (TCA) showed the best antiviral activity against EV71 at 100, 50 and 25 TCID50 with cytopathic effect less than 50% at a concentration 50, 50 and 100 μg/ml, respectively.Second, all extracts were tested for antimicrobial activity by disc diffusion method to determine the inhibition zone and by using microtitre plate-based antimicrobial assay to determine minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) against four gram positive bacteria (Staphylococcus aureus ATCC 25923, Methicillin-resistant Staphylococcus aureus DMST 20651, Staphylococcus epidermidis ATCC 12228 and Streptococcus pyogenes ATCC 19615), one gram negative bacterium (Klebsiella pneumoniae ATCC 700603) and one fungus (Candida albicans ATCC 90028). KHE showed inhibition zone against four gram positive bacteria S. aureus, methicillin-resistant S. aureus, S. epidermidis and S. pyogenes with the inhibition zone of 7.33±0.58, 7.00±0.00, 8.00±0.00 and 12.67±0.58 mm, respectively. KHA was not able to inhibit all microbes. The ethanolic extract of S. exigua (SEE) showed the highest antimicrobial activity against these five gram positive bacteria with an inhibition zone of 12.67±0.58, 13±0.00, 14.33±0.58 and 16.00±1.00 mm, respectively and showed a low inhibition zone of 8.67±0.58 mm against C. albicans. Neither ethanolic nor aqueous extracts of Kheaw-Hom remedy and its plant ingredients were able to inhibit gram negative K. pneumoniae.In addition, all extracts were tested for antimicrobial activity by microtitre plate-based antimicrobial assay to determine minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC). The results showed that KHE had antimicrobial activity against S. aureus, methicillin-resistant S. aureus, S. epidermidis and S. pyogenes with MIC values of 0.625, 0.625, 1.25 and 0.625 mg/ml and MMC values of 1.25, 0.625, 2.5 and 0.625 mg/ml, respectively but had no activity against K. pneumonia and C. albicans. The ethanolic extract of S. exigua (SEE) showed the highest antimicrobial activity against these four gram positive bacteria with MIC values of 0.005, 0.005, 0.039 and 0.019 mg/ml and MMC values of 0.005, 0.005, 0.039 and 0.019 mg/ml, respectively. Most of the aqueous extracts of Kheaw-Hom remedy and its plant ingredients had no activity against all bacteria and fungi.Third, the anti-inflammatory activities by determination of inhibitory effects on lipopolysaccharide (LPS) induced nitric oxide (NO) release from murine macrophages cell lines (RAW 264.7) were investigated. The results found that the KHA showed anti-inflammatory activity with IC50 value of 46.86±0.82 μg/ml which was higher than the KHE showed IC50 value of 59.77±3.76 μg/ml. The ethanolic extract of M. siamensis (MSE) showed the highest anti-inflammatory activity with IC50 value of 11.55±2.70 μg/ml. The aqueous extract of S. exigua (SEA) showed the highest anti-inflammatory activity with IC50 value of 3.17±0.68 μg/ml.The stability test of Kheaw-Hom extract found that the ethanolic extract of Kheaw-Hom remedy was stable in antimicrobial activity against MRSA with MIC value of 0.625 mg/ml for at least 8 months and the aqueous extract of Kheaw-Hom remedy was stable in antiviral activity against 25 TCID50 of EV71 for at least 1 year and 8 months.  Kheaw-Hom remedy was analyzed by using gas chromatography-mass spectrometry (GC-MS). There are forty-seven components found in the ethanolic extract of Kheaw-Hom remedy. The highest content was ethyl p-methoxycinnamate (18.64%) and the second highest was patchouli alcohol (16.38%).In conclusion, the aqueous extract of Kheaw-Hom remedy (KHA) showed antiviral and anti-inflammatory activities while the ethanolic extract of Kheaw-Hom remedy (KHE) showed good antimicrobial activities. The aqueous extract of T. chantrieri (TCA) showed the best antiviral activity against EV71 and the aqueous extract of S. exigua (SEA) showed the best anti-inflammatory activities. Furthermore, the ethanolic extract of M. siamensis (MSE) showed the best antimicrobial activities and had good antiviral and anti-inflammatory activities. Thus, these results support the use of Kheaw-Hom remedy to treat exanthematous fevers and skin infection complications such herpes zoster, chickenpox and hand, foot and mouth disease (HFMD) because this remedy showed antiviral properties against EV71, antimicrobial properties against microbes related to skin infection complications and anti-inflammatory activities. Moreover these results are related to Thai traditional medical practice.