Tinospora Sinensis (Lour.) Merr. Stem Modulate The TNF-Alpha Expression In HCT- 116 Tumour Cell, Besides the Inhibitory Effect on Cervical, Colon and Breast Cancer Cell Lines and Mycobacterium Tuberculosis H37Rv

Since ancient time, many countries have been practicing traditional medicines for several life threatening diseases1. In recent time, there has been remarked drug development researches based on traditional knowledge and thus, many useful explorations were conducted by scientists for identification of diverse group of medicinal plants2. Today, the co-administration of immunomodulatory agents or the use of immunoboosters are in clinical practice for treating autoimmune disorders, tubercular infections, inflammatory conditions and cancer3. Among them, cancer and multi-drug resistant tuberculosis (MDR-TB) are continuously evolving as major health burden prevailing globally4. The interleukins (IL-1, IL-10, IL-12) and tumor necrosis factor (TNF) can significantly modulate macrophage the activity, thus they can interfere in tumour growth. In human, TNF is a pleiotropic cytokine exhibit significant role in immune homeostasis, inflammatory effect and host defense. In tumour, TNF can induce apoptosis, necrosis, angiogenesis, immune cell activation, differentiation, and cell migration. TNF in combination with melphalan has already been used in treating soft tissue sarcoma and also few preclinical data suggested that TNF neutralization can fight cancer-associated complications5. Due the drug resistance of Mycobacterium tuberculosis and the reduced the immune response of host, now the disease is prevailing as multi-drug resistant (MDR), extensively resistant (XDR) and totally resistant tuberculosis (TDR-TB)6.


INTRODUCTION
Since ancient time, many countries have been practicing traditional medicines for several life threatening diseases 1 . In recent time, there has been remarked drug development researches based on traditional knowledge and thus, many useful explorations were conducted by scientists for identification of diverse group of medicinal plants 2 . Today, the co-administration of immunomodulatory agents or the use of immunoboosters are in clinical practice for treating autoimmune disorders, tubercular infections, inflammatory conditions and cancer 3 . Among them, cancer and multi-drug resistant tuberculosis (MDR-TB) are continuously evolving as major health burden prevailing globally 4 . The interleukins (IL-1, IL-10, IL-12) and tumor necrosis factor (TNF) can significantly modulate macrophage the activity, thus they can interfere in tumour growth. In human, TNF is a pleiotropic cytokine exhibit significant role in immune homeostasis, inflammatory effect and host defense. In tumour, TNF can induce apoptosis, necrosis, angiogenesis, immune cell activation, differentiation, and cell migration. TNF in combination with melphalan has already been used in treating soft tissue sarcoma and also few preclinical data suggested that TNF neutralization can fight cancer-associated complications 5 . Due the drug resistance of Mycobacterium tuberculosis and the reduced the immune response of host, now the disease is prevailing as multi-drug resistant (MDR), extensively resistant (XDR) and totally resistant tuberculosis (TDR-TB) 6 . jaundice patients 9 .Reports on antileishmanial activity and anti mycobcterial activity for TS are available 10 . There is evidence that G1-4A, a polysaccharide derived from TS inhibits Mtb survival 11,12 .

Collection and authentication of plant part
Fresh stem of Tinospora sinensis (TS) were collected from the Anantapuramu, Andhra Pradesh, India. A voucher specimen of the plant part and plant was deposited at the Biodiversity conservation division, Sri Krishnadevaraya University, Anantapuramu, AP, India (Voucher No. 55585). The collected plant material was washed with distilled water for removal of mud, dust and contaminants, and then shade dried at room temperature for 10 days. The dried stem material was blended into fine powdered. The powdered was stored in air tight container until further use.

Preparation of extracts
About 100g of TS stem powder was extracted by cold maceration technique using ethanol (95%), ethyl acetate and n-hexane. Here, TS stem powder was soaked into each solvent and was kept 7 days at room temperature (25±2 ºC) in dark room. On 7 th day, each extract was filtered using Whatman No.1 filter paper and the obtained filtrate was concentrated using rotary evaporator. All these extracts, namely ethanolic extract (ELTS), ethyl acetate extract (EATS), n-hexane (NHTS) were concentrated into semisolid mass and stored in air tight container until further use 13 .
Phytochemical analysis of extracts 100 mg of each, stem extract of TS was dissolved in 100 ml of respective mother solvent to get the concentration of 1 mg/ml. The diluted extract solution was filtered and subjected for various qualitative phytochemical tests as per the reported procedure.

Thin Layer Chromatography of extracts
The TS stem extracts were spotted on silica gel GF 254 pre-coated TLC plates. The spotted TLC Plates were developed using a pre-optimized mobile phase consisted of n-hexane and ethyl acetate (70:30 % v/v) in the saturated TLC trough chambers. After optimum development, plates were removed and air dried for 10-15 minutes. The plates detected by visual examination, UV light and iodine chamber. The appearance of colour of the spots under iodine vapour and UV light has also been considered for identification of chemical constituents. The retardation factor (R f ) values for the major spots were calculated and reported 15 .

Cell viability Assay for anticancer activity
The MTT assay was employed to determine the inhibitory effect of TS stem extracts on tumour cell proliferation. In this procedure, seed 200μl cell suspension of each (HeLa, HCT-116), MCF-7) in a 96-well plate at required cell density (20,000 cells per well) were allowed to grow for 24 hours. Then TS stem extracts (NHTS, EATS, ELTS) were added and the plates were incubated for 48 hrs at 37°C in a 5% CO 2 atmosphere. At the end of incubation period, plates were removed and added yellow coloured MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) to a final concentration of 0.5mg/mL of total volume. The plate was wrapped with aluminium foil to avoid light exposure and further incubated for 3 hours (Note: The incubation time varied for different cell lines). Then 100 μl of solubilisation solution (DMSO) was added with gentle stirring in a gyratory shaker to enhance dissolution blue MTT formazan crystals. The absorbance was observed on ELISA reader at 570nm and 630nm. The IC 50 value was determined by using linear regression equation i.e. Y = mx ± c. Here, Y = 50, m and c values were derived from the viability graph 16 .

Assessment of cell morphology: Acridine orange -Ethidium bromide staining
Acridine orange is a vital dye and will stain both live and dead cells. Acridine orange is a cell permeable nucleic acid selective dye that emits green fluorescence when bound to dsDNA. HCT-116 cells were seeded in 24 well plate at a density of 5×10 4 cell/well and incubated for 24h, then treated with test dug 17 . After 24h the acridine orangeethidium bromide working solution is prepared and followed the staining procedure to differentiate the live, apoptotic and necrotic cells in treated and untreated cells by using fluorescence microscope with excitation (488 nm ) and emission (550 nm ) at 200X magnification.

Luciferase Reporter Phage (LRP) assay for Antitubercular activity
Antitubercular activity for the extracts was studied on M. tuberculosis H 37 Rv (Mtb) using luciferase reporter phage (LRP) assay method. In each cryo vials, 10% albumin dextrose and 0.5% glycerol supplemented 350μl of G7H9 broth was taken and added aliquots of each TS stem extracts (NHTS, EATS, ELTS) to get the final concentration of 100 and 500μg/ml. One hundred microlitres of Mtb cell suspension was added to all the vials and incubated at 37 0 C for 72 hours using DMSO (1%) as solvent control. After first incubation, 50μl of high mycobacteriophage phAETRC202 and 40μl of 0.1M CaCl 2 solution was added into the test and control vials. Then the second incubated was carried out at 37 0 C for 4 hours. At the end of second incubation, 100μl from each vial was transferred to luminometer cuvette and then added 100μl of D-luciferin and then relative light unit (RLU) was measured using luminometer. The test extract showing RLU reduction by 50% or more when compared to control was considered as active extract 18 .

Flow cytometry assay for TNF-Alpha expression
Based on results of anticancer screening, NHTS was chosen for flow cytometry assay. In this, HCT-116 cells in a 6-well plate at a density of 3 x 10 5 cells/2 ml were incubated in a CO 2 incubator overnight at 37°C for 12 hours. Prior to NHTS treatment, except the untreated cell culture, all treatment cell cultures were stimulated with lipopolysaccharide (LPS) at 2 µg/ml concentration for 12 h. Cells treated with LPS were used as a negative control for anti-inflammatory marker expression whilst the cells with culture media was used as normal control. The cells treated with LPS and simvastatin was used as a positive control. Test cultures were treated with required test concentration of the NHTS and incubated for 24 h. At the end of 24 h, the medium was removed from all wells and washed with PBS, added 200 μl of trypsin-EDTA solution and then incubated at 37°C for 4 minutes. Further, 2 ml of culture medium was added and then cells were harvested directly into 12 x 75 mm polystyrene tubes. The tubes were centrifuged for five minutes at 2000 rpm at 25°C and then carefully decanted the supernatant. The cells were washed with PBS and added 0.5 mL of BD Cytofix/Cytoperm solution and waited for 10 minutes. Then, washed with 0.5% bovine serum albumin (BSA) in 1X phosphate-buffered saline (PBS) and 0.1% sodium azide and added 20 μL of FITC-Mouse Anti Human TNF Alpha antibody, mixed thoroughly and incubated for 30 minutes in the dark at room temperature (20° to 25°C). After incubation, washed with 1X PBS with 0.1% sodium azide, added 0.5 mL of PBS, mixed thoroughly, and analyze 19 .

Gas Chromatography-Mass Spectroscopy (GC-MS)
The NHTS was subjected to GC-MS analysis on the instrument GC-MS SHIMADZU GC-2010 with using short (< 5m) sin column or two column with maintains a constant mass flow rate of carrier gas (helium). Initially oven temperature was maintained at 70°C for 2.0 minutes, and the temperature was gradually increased up to 300°C at 10.0/35.0 minutes and 4.0μl of sample was injected for analysis. The flow rate of helium gas was set to 1.5 ml/min. The sample injector temperature was maintained at 260°C and the split ratio was 20 throughout the experiment periods. The ionization mass spectroscopic analysis was done with 70 eV. The mass spectra were recorded for the mass range 40-1000 m/z for about 35 minutes. Identification of compounds separated was based on their mass spectra, detected by using electronic signals 20 .

Statistical analysis
The results of all series of experiments were performed in triplicates and expressed as mean±SD (standard deviation). Cell quest software version 6.0, Graph pad Prism 8.01 was used for quantitative analysis and graphical data. The results obtained were expressed as the mean ± standard deviation. Significance was calculated using one-way ANOVA and student't' test. P<0.05 indicated a significant difference.

Phytochemical analysis of extracts
These crude extracts were resinous in nature and their % yields were 6.2, 8.9 and 8.4 % respectively for n-hexane, ethyl acetate and ethanol.
Thus the obtained extracts were tested for phytochemical tests and revealed presence of three common chemical constituents including alkaloids, glycosides and saponins (Table 1). Cell viability Assay for anticancer activity NHTS and EATS are relatively more active against HCT-116 (IC 50 : 177 µg/ml) and HeLa (IC 50 : 236 µg/ml), respectively ( Figure 2). However, ELTS was closely active on HeLa with IC 50 value of 277µg/ml as EATS ( Table 2). The IC 50 value for each extract was obtained from percentage of cell viability (Figure 1).

Assessment of cell morphology: Acridine orange -Ethidium bromide staining
The effect of test dug in HCT-116 cell line upon 24h incubation showing morphological changes like membrane blebbing, condensed nuclei and apoptotic body formation in concentration dependent manner ( Figure  3).

Luciferase Reporter Phage (LRP) assay for Antitubercular activity
The antitubercular activity of stem extracts of TS was performed on Mycobacterium tuberculosis H 37 Rv by LRP assay (Table 2). Each extract was screened at 100 and 500 µg/ml. At 100 µg/ml, both NHTS and EATS has shown % inhibition activity of 84% whereas ELTS shown 62% (Figure 4).

Flow cytometry assay for TNF-Alpha expression
The NHTS test extact showed 31.27 of mean fluorescence intensity for TNF-Alpha expression whereas standard drug simvastatin showed 26.7 mean fluorescence intensity against the control value of 7.06 ( Table  3). The concentrations used were 2µg/ml, 25µg/ml and 107.2µg/ml respectively for LPS (lipopolysachride), standard simvastatin and test extract. Histogram of the gated HCT-116 singlet distinguishes cells at the M1 and M2 phases, Here M1 refers to negative expression region and M2 refers to the positive expression region ( Figure 5).

Gas Chromatography-Mass Spectroscopy (GC-MS)
The n-hexane extracts revealed that the presence of phytochemicals such tembetarine (m/z 344.    stem decreased the expression of TNF-Alpha expression, and this is due to immunostimulant property. The interleukins (IL-1, IL-10, IL-12) and tumor necrosis factor (TNF) can significantly modulate macrophage the activity, thus they can interfere in tumour growth. In human, TNF is a pleiotropic cytokine exhibit significant role in immune homeostasis, inflammatory effect and host defence. Reported studies showed LRPbased assays are rapidly used to discover the new antitubercular drugs.
In LRP assay, drug susceptibility was assessed based on the production of photons by visible mycobacterium infected with specific report phage expressing the firefly luciferase gene. Hence, the antitubercular activity of stem extracts of TS was performed on Mycobacterium tuberculosis H 37 Rv by LRP assay. This report was concurrent with the earlier report but they have screened on dichloromethane extract. However, the relative potency of the TS stem on Mycobacterium tuberculosis is intact 24 . The staining reagent is used for easy determination among live, apoptotic and dead cells after treatment. Acridine orange identifies live cells and ethidium bromide for identification of dead cells. Acridine orange stained live cells appear green and ethidium bromide stained dead cells appear red when visualized by fluorescent microscopy. The GC-MS analyses of NHTS confirmed the presence of Berberine, palmatine, tembertarine, magniflorine, choline and tinosporin.

CONCLUSION
The n-hexane stem extract of Tinospora sinensis (NHTS) showed the significant cytotoxicity on HCT-116 tumour cells, Antitubercular activity on Mycobacterium tuberculosis H 37 Rv Tinospora sinensis stem can be used for cancer treatment which can boost the immunity in the patient along with its anticancer efficacy.

ACKNOWLEDGMENT
Authors are thankful to DST-FIST Facility of Raghavendra Institute of Pharmaceutical Education and Research, Anantapur, AP, India for the provided support in phytochemical screening. Authors are thankful to ICMR-National Institute of Research in Tuberculosis (NIRT), Chennai, Tamil Nadu, and India for providing in vitro antitubercular activity data.

FUNDING
No financial assistance received from funding agency.