## 2 Materials and Methods
### Biological Activities of Endophytic Bacteria
#### 2.4.2 Melanin Content Assay and Tyrosine Activity  
Cell culture: The B16 murine melanoma cell lines (B16F10) were collected from the Chinese Academy of Sciences, China (CAS). The cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Gibco Life Technologies, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Penicillin G (100 U/mL) and streptomycin (100 mg/mL) (Gibco-BRL, Grand Island, NY, USA) were added at 37 \({}^{\circ}\)C in a humidified atmosphere of 5% CO\({}_{2}\).  
Melanin measurement: The effects of melanin synthesis of secondary metabolites from the total extracts of bacterial endophytes isolated from the _V. anthelmintica_ stem were screened in accordance with the procedure described by [PERSON] et al. [19; 21]. B16 cells were seeded at a density of 2 \(\times\) 10\({}^{5}\) cells per well in a 6-well plate and incubated for 24 h. Then, test samples were added to the individual wells. The cells were incubated for 48 h, then washed twice with ice-cold PBS. Consequently, they were lysed at low temperature, and harvested cells were centrifuged at 8050\(\times\)\(g\) force for 20 min and were simultaneously investigated using proteinaceous supernatant; after that, the pellet was dissolved by adding 1 N sodium hydroxide and incubated at 80 \({}^{\circ}\)C for 1 h. Into a 96-well microplate was in placed 150 mL of each lysate and evaluated spectrophotometrically at 405 nm using a multi-plate reader. The protein content of each sample was calculated using a BCA Protein Assay Kit (Biomed, Beijing, China). The melanin amount was normalized to the cellular protein concentration. Melanin content was determined according to the following formula:  
\[\textit{Melanin content}(\%)=\frac{S}{B}\times 100\]  
where S is the absorbance of the cells treated by the samples and B is the absorbance of the wells containing untreated cells. Each measurement was carried out in triplicate.  
Tyrosinase activity assay: Analysis of the effects of bioactive natural products from the ethyl acetate extracts of endophytic bacteria associated with the _V. anthelmintica_ stem on tyrosinase activity was performed according to the previously described method [19; 21] with some modification. Firstly, the B16 murine melanoma cells were seeded in a 6-wellplate at a concentration of 2 \(\times\) 10\({}^{5}\) cells per well and inoculated for 24 h. Then, 50 \(\upmu\)g/mL of each bacterial extract was added to individual wells and incubated overnight. After incubation, cells were washed with ice-cold PBS twice and lysed with 1% Triton X-100 solution containing a 1% sodium deoxycholate for 30 min at \(-\)80 \({}^{\circ}\)C, and each lysate was centrifuged at 12,000\(\times\)\(g\) for 15 min. After determining and regulating the protein concentration of the supernatants, 90 \(\upmu\)L of the supernatant and 10 \(\upmu\)L of a freshly prepared substrate solution (10 \(\upmu\)M 8-MOP) were transferred in a 96-well plate and incubated at 37 \({}^{\circ}\)C for 60 min. The optical densities of samples were measured at 490 nm, and 8-methoxysolen (MOP) was used as a positive control.