To investigate the major constituents of Tinosporacordifolia Willd. growing on Mangiferaindica, and to evaluate the efficacy of their antibacterial and cytotoxicity activities.
The ethanolic stem extract of T. cordifolia was subjected to silica gel 60 column chromatography, thin layer chromatography and medium pressure liquid chromatography for isolation of the major compounds. Identification of purified compounds was achieved by spectroscopic methods.. The crude extract and purified compounds were screened for their antibacterial and cytotoxicity properties using standard procedures.
Two alkaloids were purified and identified as Magnoflorin (1) and Tembetarine (2). These compounds showed high antibacterial activity against Bacillus cereus and Staphylococcus aureus with both MIC (32-64 µg/ml) and MBC (128-256 µg/ml). The cytotoxicity activity of the purified compounds and crude extract was determined using MTT colorimetric assay against L929 and HEK293 cell lines. This showed weak cytotoxicity activity with IC50 values of 1162.24 to 2290.00 µg/ml and 1376.67 to 2585.06 µg/ml towards L929 and HEK293 cell lines, respectively.
The major compounds present in ethanolic stem extract of T. cordifolia growing on M. indica were extracted, purified and identified. This study suggests that these compounds exhibit great potential for antibacterial activity with weak cytotoxicity activity. They may be useful for their medicinal functions.
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Copyright © 2019 Thongchai Taechowisan
The authors have declared that no competing interest exists.
Tinosporacordifolia Willd. is a herbaceous vine of the family Menispermaceae. It was known in Thailand as "Boraphet" and was a glabrous climbing shrub found throughout the tropical regions of south east Asia. In Ayurveda, T. cordifolia was known as the king of medicinal plants for treating various ailments. It was an antispasmodic, antiperiodic, antipyretic, antidiabetic, anti-oxidant, anti-allergic and anti-inflammatory agent 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. The plant had been reported for intermittent fevers and infective conditions such as typhoid, malaria, filariasis, and leprosy13,14,15,16. It had anthelmintic properties17,18, and had been prescribed for urinary disorders, skin diseases, and eye diseases19,20. It was also used to treat gout and rheumatoid arthritis21,22,23, and had cardiotonic, hematinic, expectorant, antiasthmatic, and aphrodisiac actions24, and considered as the drug of choice in clearing the microcirculatory process of human body and other body channels25. It was reported that T. cordifolia growing on Azadirectaindica A. Juss had more bioactive potential than T. cordifolia and other Tinospora sp.26. There were a lot of T. cordifolia growing on Mangiferaindica in Nakhon Pathom, Thailand. There was no report on the compounds isolated from T. cordifolia growing on M. indica. The present study investigated the antibacterial and cytotoxicity activities of major compounds isolated from ethanolic stems extract of T. cordifolia growing on Mangiferaindica and determined the best concentration of the major compounds and crude extract responsible for these activities.
Materials & Methods
Plant Material and Extraction Procedure
Stems of T. cordifolia growing on M. indica were collected from the environs of Nakhon Pathom, Thailand, between September, 2017 and January, 2018. The stems were washed thoroughly 2-3 times with running water, cut into small pieces, and air dried under shade. The dried stems were then crushed in a grinder to coarse powder. Five hundred grams of powdered materials were added to 2000 ml of ethanol. The solution thus obtained was kept in an air tight flask for 24 h. The suspension was filtered using filter paper. Filtrate was evaporated at 60oC and a powdered form was obtained. The crude extract was prepared by adding methanol to obtain a stock concentration of 6 g/ml.
Isolation of the Compounds
The crude extract was dissolved in methanol to perform the bioautography assays27. The major compounds were isolated by silica gel 60 (230-400 mesh, Merck) column chromatography and eluted with chloroform : methanol (20:1, 15:1, 10:1, 7:1 and 5:1). Fractions were monitored by thin layer chromatography (TLC) (Kieselgel 60 F254, Merck), and spots were visualized under ultraviolet light and by heating silica gel plates sprayed with 10% H2SO4 in ethanol. The combined fractions were eluted with 60-80% chloroform in methanol by medium pressure liquid chromatography (400 x 40 mm column, Merck LiChroprep Si 60, 25-40 mm, UV-detection, 254nm) to afford fraction (fr.) A (54 mg), fr. B (98 mg) and fr. C (45 mg). The fr. C had no activity, and fr. A had low activity against tested microorganisms. Final purification of fr. B was achieved by preparative TLC (Si gel 60, 0.5 mm, Merck) to afford compound 1 (28 mg) from fr. B and compounds 2 (20 mg). The structures of purified compounds have been identified using NMR and mass spectral data. Optical rotations were measured on a Jasco P-1020 automatic digital polarimeter (Jasco International Co., Ltd., Tokyo, Japan). UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were recorded using a Bruker Tensor 27 FT-IR (Bruker Optics GmbH, Ettlingen, Germany) spectrophotometer with KBr pellets. NMR spectra were carried out on either a Bruker DRX-500 or an AM-400 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer with the deuterated solvent as an internal standard. ESI-MS (including High resolution electrospray ionisation mass spectra (HRESI-MS)) was performed on an API-Qstar-Pulsar i mass spectrometer (MDS Sciex, Concord, ON, Canada). The chemical structures of these compounds were identical with Magnoflorine (1) and Tembetarine (2) and shown in Figure 1.
An in vitro plate assay technique was used to test the inhibitory effects of the crude extract and purified compounds on the tested bacteria using the paper disk method according to Clinical Laboratory Standard Institute (CLSI)28. Sterile paper discs (6 mm, Whatman 2017-006) were loaded with 50 μl of two-fold dilution of 440 mg/ml of crude extract or 1 mg/ml of purified compounds. Four bacterial species were used in this study: Staphylococcus aureus ATCC 25932, Bacillus cereus ATCC 7064, Escherichia coli ATCC 10536, Salmonella typhimurium ATCC 23564 and Pseudomonas aeruginosa ATCC 27853 and methicillin-resistance Staphylococcus aureus SP6-106 (the clinical isolate). These bacteria were cultured in nutrient broth at 37°C for 24 h. Dilutions of bacterial suspensions were prepared using McFarland standard tubes (1 x 108 CFU/ml). The air-dried discs with various concentrations of the crude extract and purified compounds were placed on a lawn of bacterial spread on Muller Hinton agar. The plates were incubated at 37°C for 24 h. The diameter of the formed inhibition zones around each disc was recorded. The experiment was carried out in triplicate using gentamicin (30 unit/disk) (Oxoid, UK) as a reference for antimicrobial activity control.
Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentrations of the compound were tested against microorganisms in a 96-well microtiter plate by NCCLS microbroth dilution methods (NCCLS)29. The compound was twofold diluted from 0.5 μg/ml to 512 μg/ml, while the crude extract was twofold diluted from 0.13 mg/ml to 136.1 mg/ml, in nutrient broth supplemented with 10% glucose containing 0.01% phenol red as a colour indicator. Bacteria were adjusted to 105 CFU/ml for each microtiter plate. The microtiter plates were incubated at 37oC for 24 h. Microbial growth was determined by observing the change of colour in the wells (red to yellow when there is microbial growth). The lowest concentration that showed no change of colour was considered as the MIC, which was determined by inoculating onto nutrient agar plates 10 μl of medium from each of the wells from the MIC tests which showed no turbidity. The plates were incubated at 37°C for 24 h. Minimum bactericidal concentration (MBC) was defined as the lowest concentration of the test agent at which no microbial growth was observed on the plates.
Cytotoxicity Activity Assay
In order to evaluate the cytotoxicity activity of the crude extract and purified compounds, a cytotoxicity test was performed and the effects of the median inhibitory dose (IC50) on the murine fibroblast cell (L929) and embryonic kidney cell (HEK293) lines were assessed. These cell line were obtained from the Korean Cell Line Bank (Seoul, Korea). Different concentrations (1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 µg/ml) of the crude extract and purified compounds were prepared and used in the cytotoxicity test. To measure cytotoxicity, 5 x 104 cells were seeded in 96-well plates and incubated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum containing different concentrations of the test agents at 37oC for 24 h in 5% CO2 incubator. The wells were washed with a serum-free medium. Vehicle control groups were added with double distilled water.
In the tetrazolium salt, 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay30, yellow MTT is reduced to purple formazan in the mitochondria of viable cells. One hundred microliters of the MTT working solution (0.5 mg/ml) was added to each well and incubated at 37oC for 5 h. Next, the media were removed, wells were washed with phosphate buffer saline and 100 μl of DMSO was added to solubilize the formazan crystalline product. The absorbance was measured with a plate reader (Packard AS10000 Spectrocount, USA) at 590 nm. The production of formazan dye was proportional to the number of viable cells.
The inhibition of the cell lines cytotoxicity rates for each test agents with different concentrations was calculated according to the following equation:
%Inhibition = 100 – ((Abssample - Absblank) / (Abscontrol - Absblank)) x 100
Where Abssample is the absorbance of the test agent and Abscontrol is the absorbance of the control reaction (containing all reagents except the test agent). The %inhibition was plotted against sample concentration, and a linear regression curve was established in order to calculate the IC50. Tests were carried out in triplicate. Correlation coefficients were optimized.
Ethanolic extract from the stems of T. cordifolia was purified by column chromatography and TLC. In the active fraction, two major compounds were isolated and identified as follows.
Compound 1: Magnoflorine (1): C20H24NO4; white amorphous powder; UV(MeOH) lmax(log ε) = 225, 273, 309 nm; 1H-NMR (d6-DMSO): G(ppm) = 2.95 (s, 3H); 3.40 (s, 3H); 3.83 (s, 3H); 3.89 (s, 3H); 4.50 (m, 1H); 7.03 (b.s., 3H); 9.9 (m, 2H). 13C-NMR (12C-d8-DMS0): 142.2 (1); 120.9 (1a); 120.0 (1b); 148.8 (2); 110.4 (3); 120.2 (3a); 23.2 (4); 60.0 (5); 68.2 (6a);29.8(7); 125.8(7a); 119.4(8); 111.4(9); 149.3 (10); 141.6(11); 119.7(11a);42.9(N-CH3);53.1 (N-CH,); 56.1 (2-OCH3); 55.9 (10-OCH,); MS (70eV, m/e (%)): 341(1); 327(0.5); 284(0.5); 283(0.4); 270(2); 256(3); 142(10); 128(50); 127(20); 58(100).
Compound 2: Tembetarine (2): C20H26NO4; white amorphous powder; UV(MeOH) lmax (log ε) = 210(4.45); 228sh (4.2); 284 (3.8). 'H-NMR (d6-DMSO): G(ppm) = 3.16 (s, 3H); 3.36 (s, 3H); 3.80 (s, 6H); 4.73 (m, 1H); 6.00 (s, 1H); 6.45-7.10 (m, 4H); 8.99 (s, 2H). 13C-NMR (12C-d6-DMS0): 71.1(1); 50.3 (N-CH,); 54.5(N-CH3); 50.5(3); 22.9(4); 123.2(4a); 111.6(5); 146.6(6); 146.4(7); 114.7(8); 119.0(8a); 36.7~; 128.5(1'); 116.3(2'); 144.6(3'); 147.7(4'); 112.1(5'); 120.2(6'); 55.9(6-OCH3); 55.6(4'-OCH3). MS (70 eV; m/e (%)): 343(6); 206(3); 192(100); 177(20); 149(8); 148(7); 142(17); 128(7); 127(8); 58(75). Compounds 1 and 2 were identified as magnoflorine (1) and tembetarine (2), respectively. Their 'H- and 13C-NMR spectral data were identical with those of magnoflorine and tembetarine previously reported by Pachaly and Schneider31.
The crude extract from the stems of T. cordifolia showed a dark brown color. The crude extract yield was 12.0 g/kg while the percentage yields of the purified compounds 1 and 2 were about 0.47% and 0.33% (w/w), respectively. The antibacterial activity of the crude extract and purified compounds is summarized in Table 1. Various concentrations of crude extract and purified compounds were tested using agar disc diffusion assay. A zone of inhibition >8 mm in diameter was interpreted as sensitive. All of the susceptible strains were sensitive to the crude extract at 30 mg/disc. The crude extract showed the highest activity against B. cereus ATCC 7064, MRSA SP6-106and S. aureus ATCC 25932 at 30 mg/disc with the average zones of inhibition being 14.33 ± 2.25 mm, 15.73 ± 2.77 mm and 18.28 ± 3.68 mm, respectively. However, this crude extract showed low activity against E. coli ATCC 10536 and S. typhimurium ATCC 3564 at 30 mg/disc with the average zones of inhibition 8.33 ± 2.88 mm and 8.43 ± 2.17 mm, respectively, and also showed moderate activity against P. aeruginosa ATCC 27853 at 30 mg/disc with the average zones of inhibition 10.27 ± 2.65 mm. Compounds 1 and 2 showed the highest activity against B. cereus ATCC 7064, MRSA SP6-106and S. aureus ATCC 25932 at 50 µg/disc with the zones of inhibition ranging from 16.34 ± 4.53 mm to 19.67 ± 3.68 mm. They also showed moderate activity against P. aeruginosa ATCC 27853, E. coli ATCC 10536 and S. typhimurium ATCC 3564 at 50 µg/disc with the zones of inhibition ranging from 8.66 ± 2.31 mm to 10.50 ± 2.83 mm. Sensitive results were not obtained with discs containing 3.75-7.5 mg/disc of the crude extract and 1-5 µg/disc of compounds 1 and 2 against E. coli ATCC 10536 and S. typhimurium ATCC 3564, and 7.5 mg/disc of the crude extract and 1 µg/disc of compounds 1 and 2 against P. aeruginosa ATCC 27853.Table 1. Antibacterial activity of the crude extract and purified compounds on the tested microorganisms.
|Tested agents/concentrations||Diameters of inhibition zones on tested microorganisms (mm)|
|Crude extract||3.7 mg/disc||7.33 ± 1.67||7.84 ± 1.72||NZ||NZ||NZ||7.71 ± 1.84|
|7.5 mg/disc||8.50 ± 1.33||8.86 ± 1.64||NZ||NZ||7.62 ± 1.56||8.92 ± 2.53|
|15 mg/disc||10.28 ± 2.67||13.16 ± 2.54||7.72 ± 1.77||7.35 ± 1.24||8.65 ± 2.51||12.62 ± 2.86|
|30 mg/disc||14.33 ± 2.25||18.28 ± 3.68||8.33 ± 2.88||8.43 ± 2.17||10.27 ± 2.65||15.73 ± 2.77|
|Compound 1||1 mg/disc||8.22 ± 1.14||8.57 ± 2.13||NZ||NZ||NZ||8.74 ± 1.62|
|5 mg/disc||8.87 ± 1.67||9.20 ± 2.38||NZ||NZ||8.24 ± 2.52||9.16 ± 2.33|
|10 mg/disc||13.21 ± 5.08||14.67 ± 3.92||8.34 ± 2.16||8.30 ± 2.62||9.11 ± 2.77||14.10 ± 2.36|
|50 mg/disc||16.34 ± 4.53||18.88 ± 3.97||8.66 ± 2.31||8.72 ± 2.35||10.50 ± 2.83||18.21 ± 3.33|
|Compound 2||1 mg/disc||7.14 ± 1.77||8.64 ± 2.65||NZ||NZ||NZ||8.56 ± 2.21|
|5 mg/disc||8.38 ± 2.83||9.50 ± 2.76||NZ||NZ||8.08 ± 2.85||8.85 ± 2.38|
|10 mg/disc||12.50 ± 3.44||13.50 ± 3.33||8.21 ± 2.37||8.16 ± 2.13||8.88 ± 2.84||12.54 ± 2.82|
|50 mg/disc||16.35 ± 3.88||19.67 ± 3.68||8.72 ± 2.63||8.81 ± 2.47||9.50 ± 2.77||18.51 ± 3.55|
|Gentamicin||10 mg/disc||23.76 ± 1.74||22.88 ± 1.33||21.45 ± 1.61||20.74 ± 1.84||21.35 ± 1.46||23.58 ± 1.66|
Adopting a classification based on MIC values proposed by Kuete32, and Kuete and Efferth33, the antibacterial activity of a plant extract is considered significant when the MICs are below 100 µg/ml, moderate when 100 ≤ MIC ≤ 512 µg/ml, and weak if MIC > 512 µg/ml. Consequently, where the activity of the crude extract showed MIC values greater than 512 µg/ml, it was therefore considered a weak inhibitor against all the test microorganisms. Compounds 1 and 2 showed the lowest MIC (32 µg/ml) against B. cereus ATCC 7064 (Table 2). These were followed by the MIC values (64 µg/ml) against MRSA SP6-106 and S. aureus ATCC 25932. Compounds 1 and 2 had high MIC values (512 µg/ml) against P. aeruginosa ATCC 27853, E. coli ATCC 10536 and S. typhimurium ATCC 3564. Compounds 1 and 2 showed the lowest MBC (128-256 µg/ml) against B. cereus ATCC 7064, MRSA SP6-106 and S. aureus ATCC 25932 whereas these compounds had high MBC values (>512 µg/ml) against P. aeruginosa ATCC 27853, E. coli ATCC 10536and S. typhimurium ATCC 3564. The crude extract had no inhibitory activity in MBC against P. aeruginosa ATCC 27853, E. coli ATCC 10536and S. typhimurium ATCC 3564.Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of crude extract, purified compounds on tested microorganisms.
|Tested microorganisms||Antibacterial activity of the tested agents|
|Crude extract (mg/ml)||Compound 1||Compound 2||Chloramphenicol (mg/ml)|
To evaluate the cytotoxicity activity of the crude extract and purified compounds against L929 and HEK293, the cell lines were incubated with different doses of two-fold dilution (1-512 µg/ml) of the crude extract and purified compounds. After 24 h of incubation, cell viability was determined by MTT assay. The crude extract and purified compounds induced cell cytotoxicity in a concentration-dependent manner. The corresponding IC50 was calculated, and the results are presented in Table 3. The cytotoxicity activity of the crude extract and purified compounds was observed and showed weak cytotoxicity activity with IC50 values of 1162.24 to 2290.00 µg/ml and 1376.67 to 2585.06 µg/ml towards L929 and HEK293 cell lines, respectively.Table 3. IC50 of the crude extract, purified compounds against normal cell lines after 24 h using the MTT assay.
|IC50a values of crude extract, purified compounds on tested cell lines (g/ml)|
|Test microorganisms||L929b cells||HEK293c cells|
Medicinal properties of plant derived compounds are known to show curative activity against several bacteria and it is not surprising that the medicinal plant extracts are used traditionally by herbalists to treat bacteria related ill-health.
T. cordifolia also exerted considerable antibacterial effect against tested pathogens. This plant has been extensively subjected to chemical investigations, and a number of chemical constituents belonging to different groups such as trepenoids, alkaloids, diterpenoid lactones, sesquiterpenoids, lignans, flavonoids, tannins, cardiac glycosides, steroids have been reported, which may account for the antimicrobial property of these agents 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. Our research findings regarding the major compounds of T. cordifolia from Nakhon Pathom, Thailand, differ from previous reports in the literature regarding T. cordifolia from other geographical regions. Bisset and Nwaiwu53 reported that the major quaternary alkaloids in Tinospora species were generally the protoberberine bases berberine and palmatine. Nagaprashanthi et al.54 found that the hydroalcoholic extract of T. cordifolia grew over Azadirachtaindica (neem plants) has potential antimicrobial activity similar to Azadirachtaindica, and also has higher potential antimicrobial activity than the hydroalcoholic extract of T. cordifolia climbing on fencing. This may explain why the host plants (T. cordifolia) will acquire the medicinal properties when they survive on neem plants and their extracts contain more of the active compounds. In this study, therefore, the extract of T. cordifolia growing on M. indica from Nakhon Pathom, Thailand, was found to have a significantly different chemical composition from the extract of T. cordifolia from other geographical locations. This property could be attributed to exchange of bioactive constituents from M. indica to T. cordifolia, but further studies are warranted to elucidate the exact mechanisms responsible for the observed bioactive compounds. In addition, variations in the chemical composition of the extracts are known to differ considerably due to the existence of different subspecies. They might also be attributable to other factors such as climate, different regional geographic and seasonal conditions, metabolism of plants, stage of maturity and extraction conditions55. Alkaloids like berberine, palmatine, tembetarine, magnoflorine, choline, tinosporin, columbin, isocolumbin, tetrahydropalmatine have been isolated from stem and root extracts of this plant 53, 54, 55, 56, 57, 58, 59. They present numerous biological activities such as being emetic, anticholinergic, antitumor, diuretic, sympathomimetic, antiviral, antihypertensive, analgesic, antidepressant, muscle relaxant, anti-inflammatory, antimicrobial, and antiulcer60. The alkaloids have proton-accepting nitrogen atom and one or more proton-donating amine hydrogen atoms, which form hydrogen bonds with proteins, enzymes, and receptors. Furthermore, they, generally, have functional groups such as phenolic hydroxyl. The later might explain the exceptional bioactivity of the alkaloids61. In this study, major bioactive compounds; magnoflorine and tembetarine were isolated from T. cordifolia, and have antibacterial activity especially on Gram-positive bacteria. The biological activities, such as antioxidant, anti-a-glucosidase, a-tyrosinase inhibitory, anti-inflammatory, and anticancer activities, of magnoflorine have been reported. Magnoflorine from Aristolochia debilis stems showed significant antioxidant activity as a DPPH free radical scavenger, considerable a-tyrosinase inhibitory effect and also showed considerable anti-inflammatory activity in high dosage62. Hung et al.63 also have reported that, magnoflorine plays a role in protecting high-density lopoprotein (HDL) under oxidative stress. Patel and Mishra64 indicated that magnoflorine from T. cordifolia stems inhibited a-glucosidase activity. For anticancer activity, the cytotoxic effect of T. cordifolia stem extracts against cancer prostate (PC-3), colon (Colo-205, HCT-116), lung (A546, NCIH322) and breast cancer (T47D) cell lines have been reported 65, and magnoflorine has shown selected cytotoxicities against the murine leukemia (P388) cell line66. The antibacterial properties of T. cordifolia have been investigated by researchers world wide54,67,68,69. Kumar et al.70 have reported that, the crude extract from T. cordifolia leaves had antibacterial effect on E. coli. However this research found that the crude extract from T. cordifolia stems had low antibacterial activity against this microorganism. Variations in the chemical composition of the compounds are known to differ considerably not only due to the existence of different part of plants or subspecies, but might also be attributed to other factors such as climatic, geographic and seasonal condition of the regions, metabolism of plants, stage of maturity and extraction conditions55. With regard to the purified compounds, magnoflorine and tembetarine exhibited antibacterial activity that varied between bacterial species (MIC = 32 – 512 µg/ml). This differs from the report of Mushtaq et al.71. They found that magnoflorine isolated from Aquilegia fragrans exhibited weak antibacterial activity against various mastitis pathogens such as S. aureus, and Staphylococcus equorum with MIC values of 500 µg/ml.
Tinospora and Aristolochiaplant overdoses may have serious renal side-effects. Testing the cytotoxicity of the crude extract and purified compounds was carried out on L929 and HEK293 cells. The crude extract and purified compounds showed no toxicity on both cells even at high dosage. That means, they had no cytotoxicity on L929 and HEK293 cells. This was similar to the findings of Li and Wang62, that magnoflorine had no toxicity on HEK293 and HT-29 cells at the concentration of 400 µg/ml.
The results obtained in this study thus suggest that the major bioactive compounds (compound 1; magnoflorine and compound 2; tembetarine) isolated from T. cordifolia have antibacterial activity especially on Gram-positive bacteria. These compounds showed bactericidal effects against B. cereus ATCC 7064, MRSA SP6-106 and S. aureus ATCC 25932 with both MIC and MBC at a concentration of 32-64 µg/ml and 128-256 µg/ml, respectively. Compounds 1 and 2 showed weak cytotoxicity activity against normal fibroblast L929 and embryonic kidney HEK293 cells and may be useful for their medicinal functions.
This work was supported by the Department of Microbiology, Faculty of Science, Silpakorn University, Thailand.