Antiviral activity against herpes simplex virus type 2 of Kheaw-Hom remedy extract and its plant ingredients

Herpes simplex virus type 2 (HSV-2) occurs on the skin as a common infection to patients of all genders and all ages. Acyclovir (Zovirax), famciclovir (Famvir), and valacyclovir (Valtrex) are medications of choice to be used in treating HSV-2 infection. However, resistance to acyclovir is frequently found in hospitals. Interestingly in alternative drugs, including natural products and herbal medicine, are increased recently. Kheaw-Hom as a Thai traditional remedy is commonly used to relieve fever, treat aphthous ulcers, and regarding to treat symptoms from measles and chickenpox, but there are a few report for treating HSV-2. It consists of 18 herbs: Bua-luang (Nelumbo nucifera Gaertn.), Bun-nak (Mesua ferrea Linn.), Chan-dang (Dracaena cochinchinensis), Chan-thet (Myristica fragrans Houtt.), Faek-hom (Vetiveria zizanioides (L.) Nash ex Small), Ma-has-sa-dam (Cyathea gigantea Holtt.), Mak-mia (Cordyline fruticose (L.) A.Chev (Green leaves)), Mak-phu (Cordyline fruticosa (L.) A.Chev (Red leaves)), Nae-ra-phu-sri (Tacca chantrieri Andre.), Phak-kra-chom (Limnophila rugosa (Roth) Merr.), Phi-kul (Mimusops elengi Linn.), Phim-sen-ton (Pogostemon cablin (Blanco) Benth.), Phit-sa-nat (Sophora exigua Craib.), Proh-hom (Kaempferia galanga Linn.), San-phra-hom (Eupatorium stoechadosmum Hance.), Sa-ra phi (Mammea siamensis Kosterm.), Wan-kep-rat (Angiopteris evecta (G.Forst) Hoffm.), and Wan-ron-thong (Globba malaccensis Ridl.). The aims of this study were to investigate antiviral activity against HSV-2 by using aqueous and ethanolic extracts of Khaew-Hom, its plant ingredients, and its fractions. In vitro cytotoxic and antiviral activities of aqueous and ethanolic extracts of Kheaw-Hom, its plant ingredients and fractions revealed high potency exhibition against HSV-2. First, cytotoxicity of all extracts was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. These extracts were investigated for antiviral activity by plaque reduction assay, including HSV-2 preincubation, pretreatment, and posttreatment by plaque reduction assay. Then, the most effective extract of Kheaw-Hom was isolated into five fractions by column chromatography to test all experiments. Finally, all extracts were screened for chemical compounds by thin layer chromatography (TLC).Results were found that the aqueous and ethanolic extracts showed no toxicity to Vero cells. The aqueous extract of Kheaw-Hom exhibited against HSV-2 with IC50 values of 94.24±1.26 µg/mL and ethanolic extract showed no inhibition of HSV-2 by pre-incubation. In addition, aqueous extract of 18 plant ingredients in Khaew-Hom exhibited potent antiviral activity against HSV-2 by pre-incubation: C. gigantea (IC50 = 10.84±2.68 µg/mL), P. cablin (IC50 = 30.72±7.66 µg/mL), N. nucifera (IC50 = 37.08±9.72 µg/mL), M. ferrea (IC50 = 39.23±4.29 µg/mL), M. elengi (IC50 = 60.19±9.08 µg/mL), D.cochinchinensis (IC50 = 61.83±7.37 µg/mL) and M. siamensis (IC50 = 67.1±13.28 µg/mL). Fractionally, four examples of potent antiviral activity were seen against HSV-2 by pre-incubation: FA2 (IC50 = 18.86±6.02 µg/mL), FA3 (IC50 = 20.44±5.09 µg/mL), FA4 (IC50 = 31.41±6.71µg/mL), and FA5 (IC50 = 85.43±7.69 µg/mL). These extracts exhibited direct inactivation virus effect against HSV-2. Acyclovir as a positive control exhibited against HSV-2 with IC50 values of 11.61±2.71µg/mL by pre-incubation. In pre-treatment and post-treatment, all extracts had no HSV-2 inhibition. The preliminary phytochemical investigation of Kheaw-Hom aqueous extract, its plant ingredients, and its fractions was performed using the thin layer chromatography technique. The results indicated that fraction 2 and fraction 3 consist of orange and purple spots that were not found in Kheaw-Hom aqueous extract and plant ingredients. Thus, the synergistic effect of phytochemicals may cause new compounds that were found in fractions. These results suggest that the aqueous extract of Kheaw-Hom, 7 of 18 plant ingredients and fractions 2-5 showed potent antiviral activity by pre-incubation. The mechanism of aqueous extract may be considered for antiviral activity by direct contact. Therefore, fractions should be considered to isolate pure compounds to identify a Kheaw-Hom marker related to traditional Thai medical practice. Kheaw-Hom may be developed as a topical drug suitable for treating HSV-2.