Experiment study on Ma-Xing-Shi-Gan-Tang (MXSGT) and its decomposed recipes on anti-influenza virus A

Ancient China was an East Asian country with large population. As we review the history of China, large scale epidemic like Black Death in Europe rarely occurs. It can be seen that the Traditional Chinese Medicine (TCM) make great effort to antivirus and anti-infection treatment. When the SARS broke out in China in 2003, all medical drugs cannot to prevent or treat it. Preventive with TCM and strict disinfection help control the extension of SARS in short time.The using of TCM in clinical must abide by a most important principle that called principal, assistant, complement and guide. We investigate a famous complex recipe Ma Xing Shi Gan Tang (MXSGT) which is record by well-known clinical textbook "Treatise on Cold-induced Febride Diseases". In order to exploring prescription principle of MXSGT, We examined its decomposed recipes on anti-influenza virus A in vitro. MXSGT was disassembled into 15 recipes according to the design of composing prescriptions. Cell culture technique and MTT test were used to determine the effects of MXSGT and its decomposed recipes on MDCK cells infected by influenza virus A.


Introduction
The using of Traditional Chinese Herbs in traditional medicine clinic based on unique principle---principal, assistant, complement and guide from the emergence of the earliest medicine guide book Shen Nong Materia Medica two thousand years ago to today. Ma Xing Shi Gan Tang is a most classical complex prescription derived from the well-known clinical textbook Treatise on Cold-induced Febride Diseases which was compiled by the most famous of Traditional Chinese physician Zhang Zhongjing.
This complex prescription is composed of Chinese Ephedra Herb (Ma Huang), Bitter Almond (Xing Xen), Gypsum (Shi Gao) and Liquorice Root (Gan Cao) and act as principal, assistant, complement and guide correspondingly. The drug dose of four medicines is 6g, 9g, 24g and 6g recorded by tradition medicine book. It has the pharmacological action of relieving fever, anti-inflammatory, antivirus, relieving cough and so on, it is widely used in the clinic for thousands of years with an excellent safety record [1] . The MXSGT has been reported inhibitory effects on influenza A virus in vitro and its effect may interfering influenza virus A absorption and protecting the cells infected by influenza virus A [2][3] . With regard to the inhibitory on influenza A virus of decomposed recipes, thus far there has not been reported.

Decomposed formulas
Ma Xing Shi Gan Tang prescription was disassembled into 15 recipes according to the design of decomposed recipes (Tab 1) and every recipe was examined inhibitory effects on influenza A virus in vitro.

Extraction
According to the application of prescription, all the decoctions were prepared according to the list of decomposed recipe. All materials were cut into pieces and refluxed for 1.5 h with 75% EtOH three times. The extract was filtered and concentrated under reduced pressure. The samples saved in cryopreservation. MDCK cells were used as host cells. They were grown in Eagle's minimum essential medium (EMEM) which contain 10% fetal bovine serum (FBS, Gibco), penicillin G (100 U/mL) and streptomycin (100 g/mL). The cells were maintained in a humidified atmosphere containing 5 % CO 2 at the temperature of 37 .

Toxicity measurements
MDCK cells were seeded into 96-well culture plates at a density of 1×10 4 cells per well. Cells were incubated for 24 h until 90% confluency. Samples were dissolved in DMSO and then diluted to 400 g/mL, 200 g/mL, 100 g/mL, 50 g/mL and 25 g/mL, respectively. These dilutions were incubated with monolayer MDCK cells at the temperature of 37 in 5% CO 2 for 72 h. The growth of cells was detected by modified MTT assay. Cell viability was calculated according to the following formula: Cell viability (%) = (OD Sample / OD normal control) ×100% TC 0 is the concentration at which the cell viability of the sample >=90%

In vitro antiviral evaluation
The antiviral effect of the samples on influenza virus was measured by inhibition of viral cytopathic effect (CPE). MDCK cells were seeded into 96-well culture plates at a density of 1×10 4 cells per well and incubated for 24h until 90% confluency.  Cao recipe has the same inhibition effect as MSXGT at 200 g/ml.

Discussion
Thus far, there is only a single report on the anti-influenza A virus activity of MXSGT decomposed recipe [4] . According to the results of toxicity experiments, it shows that complex prescription may imply low toxicity in view of the TC 0 of MXSGT is higher