Background
Methods
Plant material
Preparation of plant extracts and fractionation
Physicochemical analysis
Quantification of polyphenols and antioxidants
Cell cultures
Biological activity of RHTR in colon cancer and normal cells
Selectivity index (SI) and median lethal dose (LD50)
Measurement of the cell cycle
Genotoxic analysis by the comet assay
ROS-intracellular quantification by CM-H2DCFDA
Evaluation of morphology and cell death
Experimental animals
Acute toxicity studies
Determination of biochemical parameters and hematic biometry
Anatomical and histological assessments
Phytochemical characterization of RHTR by UPLC-MSE
Statistical analysis
Results
Physicochemical analysis of RHTR
Composition | Quantity |
---|---|
Carbohydrates | 309.3 ± 27.5 |
Amino acids | 15.9 ± 3.7 |
Proteins | 21.1 ± 3.3 |
Lipids | 4.5 × 10−4 ± 2.1 × 10− 4 |
% Humidity | 7.9 ± 2.1 |
% Total ash | 3.5 ± 0.04 |
% Soluble ash H2O | 2.8 ± 0.5 |
% Insoluble ash HCl | 1.0 ± 0.5 |
pH | 4.4 ± 0.3 |
Contents of polyphenols and antioxidants in AE and FF from RHTR
Samples | AE | FF |
---|---|---|
Weight (mg) | 590 ± 49.5 | 22 ± 4.9 |
Polyphenols (Ga Eq) | 94.14 ± 7.8 | 16.38 ± 3.8 |
Flavonoids (Que Eq) | 84.56 ± 3.6 | 27.4 ± 5.7 |
Anthocyanins (Cya Eq) | 9.98 ± 1.5 | 0.043 ± 0.01 |
Antioxidants (Trolox Eq) | 197.5 ± 11.3 | 13.2 ± 0.09 |
AC50 | 0.042 | 0.105 |
TAC50 | 1 | 41 |
% DPPH Inhibition | 86.07 | 14.10 |
AEI | ND | ND |
Antioxidant activity | Very high | Low |
Biological activity of AE and FF from RHTR in cancer and normal cells
AE | FF | Vincristine | IX PBS | |
---|---|---|---|---|
Cells
| In vitro studies: IC50, SI and LD50 | |||
IC50 CACO-2 | 5 (High) | 18 (High) | 25 (High) | > 800 (Very low) |
IC50 BEAS-2B | 800 (Very low) | 600 (Very low) | 25 (High) | > 800 (Very low) |
IC50 CHO-K1 | 600 (Very low) | 600 (Very low) | 57 (medium) | > 800 (Very low) |
SI | 140 +++ | 33.3 ++ | 1.64 + | ND |
LD50 [Theoretical] | 1141.5 | 1209 | 350 | ND |
In vivo studies: acute toxicity | ||||
Sign toxicity | None | None | ND | None |
Survivors (%) | 100 | 100 | ND | 100 |
Final weight (g) | 28.6 ± 2.4 | 28.2 ± 2.4 | ND | 26.5 ± 2.3 |
Acute toxicity evaluation in BALB/c mice with AE and FF from RHTR
Time | Treatment | Measurement | Kidney | Heart | Lung | Spleen | Liver |
---|---|---|---|---|---|---|---|
24 h | AE | W | 0.36 ± 0.04 (1.4%) | 0.13 ± 0.02 (0.5%) | 0.22 ± 0.02 (0.8%) | 0.11 ± 0.01 (0.4%) | 1.44 ± 0.2 (5.5%) |
D | 9.23 ± 0.5 | 8.8 ± 0.45 * | 11.04 ± 1.4 | 14.5 ± 1.16 | 29.6 ± 0.96 | ||
FF | W | 0.37 ± 0.04 (1.5%) | 0.12 ± 0.01 (0.5%) | 0.22 ± 0.05 (0.9%) | 0.10 ± 0.03 * (0.4%) | 1.36 ± 0.3 (5.4%) | |
D | 10.17 ± 0.8 | 8.42 ± 0.64 | 11.6 ± 0.42 | 15.1 ± 1.8 | 26.1 ± 2.96 | ||
1X PBS | W | 0.35 ± 0.06 (1.4%) | 0.12 ± 0.02 (0.5%) | 0.23 ± 0.03 (0.9%) | 0.13 ± 0.02 (0.5%) | 1.3 ± 0.21 (5.4%) | |
D | 10.06 ± 0.5 | 8.19 ± 1.2 | 11.94 ± 1.1 | 14.1 ± 2.12 | 27.9 ± 1.2 | ||
14 days | AE | W | 0.37 ± 0.01 (1.3%) | 0.16 ± 0.02 (0.5%) | 0.26 ± 0.02 (0.9%) | 0.11 ± 0.01 (0.4%) | 1.29 ± 0.09 (4.5%) |
D | 10.3 ± 0.55 | 9.5 ± 1.7 | 13.74 ± 0.9 | 17.4 ± 1.15 | 28.6 ± 4.83 | ||
FF | W | 0.22 ± 0.14 (0.8%) | 0.17 ± 0.02 (0.6%) | 0.28 ± 0.05 (0.9%) | 0.14 ± 0.01 * (0.5%) | 1.48 ± 0.15 (5.3%) | |
D | 10.34 ± 0.4 | 9.35 ± 0.14 | 13.93 ± 1.7 | 17.01 ± 0.6 | 30.5 ± 0.86 | ||
1X PBS | W | 0.35 ± 0.05 (1.3%) | 0.16 ± 0.03 (0.6%) | 0.21 ± 0.01 (0.8%) | 0.12 ± 0.01 (0.5%) | 1.35 ± 0.12 (5.1%) | |
D | 10.8 ± 0.6 | 10.03 ± 0.8 | 14.6 ± 2.95 | 15.2 ± 1.4 | 31.8 ± 3.3 |
Time | 24 h. | 14 days | Reference range | ||||
---|---|---|---|---|---|---|---|
Treatment | AE | FF | 1X PBS | AE | FF | 1X PBS | (Midrange) |
BIOCHEMICAL PARAMETERS | |||||||
Glucose (mg/dL) | 118.7 ± 13.8 | 113.7 ± 17.5 | 138.7 ± 17.9 | 130.2 ± 6.9 | 127.7 ± 15.4 | 141.5 ± 10.4 | 63–176 (89) |
Triglycerides (mg/dL) | 70.13 ± 11.3 | 74.6 ± 7.4 | 81.4 ± 8.6 |
121.8 ± 17.2
| 112.75 ± 19.8 | 98.5 ± 7.3 | 55–115 (85) |
Cholesterol (mg/dL) | 76.3 ± 8.5 | 77.5 ± 14.4 | 75 ± 8.2 | 61 ± 12.7 | 55 ± 5.8 | 65 ± 2.8 | 26–82 (64) |
Protein (g/dL) | 5.4 ± 0.5 | 5.5 ± 0.6 | 5.4 ± 0.25 | 5.7 ± 0.63 | 5.2 ± 0.5 | 5.8 ± 0.82 | 4–8.6 (6.2) |
AST (TGO) (U/L) | 205 ± 45.3 * | 152.5 ± 8.7 | 127.5 ± 19.4 | 132 ± 19.7 | 165 ± 45.5 | 161.8 ± 47.5 | 55–251 (139) |
ALT (TGP) (U/L) | 55.9 ± 8.8 |
78.7 ± 0.3 *
| 52.9 ± 6.2 | 64.4 ± 1.4 | 74.7 ± 16.1 | 70.7 ± 5.8 | 17–77 (47) |
LDH (U/L) |
1234 ± 221
|
954 ± 281
|
1330 ± 274
|
1122 ± 201
|
980 ± 286
|
997.3 ± 70.3
| 149–215 (182) |
Creatinine (mg/dL) | 0.62 ± 0.2 | 0.5 ± 0.01 | 0.5 ± 0.01 | 0.5 ± 0.01 | 0.5 ± 0.01 | 0.46 ± 0.09 | 0.2–0.9 (0.5) |
Urea (mg/dL) | 60 ± 5.8 | 56.2 ± 4.8 | 53.7 ± 6.3 | 65 ± 4.1 | 57.5 ± 6.5 | 56.7 ± 7.2 | 46.9–73 (60.1) |
HEMATOLOGICAL PARAMETERS | |||||||
Hemoglobin (g/dL) | 15.16 ± 1.09 | 13.5 ± 0.36 | 14.76 ± 0.90 | 16 ± 1.05 | 13.23 ± 1.8 | 14.13 ± 0.20 | 10–17 (13.1) |
Hematocrit (%) | 47.2 ± 4.07 | 39.33 ± 3.40 | 43.93 ± 2.83 | 49.5 ± 3.99 | 38.8 ± 5.63 | 43.7 ± 0.55 | 39–49 (40.4) |
Erythrocytes (X106/mm3) | 9.69 ± 1.16 |
7.34 ± 0.95
| 9.27 ± 0.97 | 9.92 ± 0.85 |
7.91 ± 1.13
| 8.84 ± 0.43 | 8.3 |
MCHC (%) | 32.06 ± 0.66 |
36.36 ± 4.56
| 33.63 ± 1.04 | 32.3 ± 0.52 |
36.26 ± 2.57
| 32.3 ± 0.72 | 32.3 |
MCH (pg) | 15.66 ± 0.8 |
14.63 ± 3.68
| 15.96 ± 0.96 | 16.1 ± 0.34 |
14.3 ± 2.4
| 15.96 ± 0.86 | 15.9 |
MCV (μL) | 48.9 ± 1.6 | 47.26 ± 11.49 | 47.5 ± 1.83 | 49.9 ± 0.3 | 48.9 ± 1.95 | 49.5 ± 1.83 | 49.1 |
Leukocytes (× 100/mm3) | 5000 ± 953.9 |
4233.3 ± 1404.7
| 5666.66 ± 808.29 |
4466.66 ± 929.15
|
4666.6 ± 1242.3
| 5033.33 ± 1227.8 | 5–12 (6.33) |
RDW-CV | 15 ± 1.17 | 18.7 ± 1.56 | 17.96 ± 1.11 | 13.73 ± 0.57 * | 16.33 ± 0.97 | 16.16 ± 1.20 | |
RDW-SD | 24.26 ± 3.18 * | 32.63 ± 18.9 | 33.2 ± 5.32 | 22.8 ± 0.51* | 26.13 ± 1.5 | 25.73 ± 1.38 | |
Platelets (/μL) |
437,333 ± 94,214
|
558,666.6 ± 230,048.5
|
627,000 ± 95,859.2
|
368,000 ± 46,130
|
425,333.3 ± 325,423
|
572,666 ± 158,190
| 116 × 103 |
MPV (fL) | 7.6 ± 0.2 | 7.5 ± 2.4 | 8.16 ± 0.87 | 7.56 ± 0.15 | 9.43 ± 0.49 * | 7.6 ± 0.3 | |
PDW (%) | 14 ± 0.1 | 14.6 ± 4.5 | 14.96 ± 2.32 | 14.06 ± 0.15 | 13.03 ± 2.8 | 14 ± 0.26 | |
PCT (%) | 0.33 ± 0.07 * | 0.27 ± 0.05 * | 0.51 ± 0.12 | 0.277 ± 0.03 * | 0.585 ± 0.12 | 0.432 ± 0.10 | |
Lymphocytes (%) | 67.33 ± 10.06 * | 74 ± 7.21 | 82 ± 2 | 65 ± 13.4 | 63.3 ± 4.16 | 68 ± 20.7 | 35–90 |
Monocytes (%) | 1.33 ± 1.15 | 0 | 0.33 ± 0.57 | 0.33 ± 0.57 | 0 | 0 | 0–3 |
Eosinophils (%) | 2 | 0 | 0 | 0 | 0 | 0 | 0–7 |
Basophils (%) | 0 | 0 | 0 | 0 | 0 | 0 | 0–1 |
Segmented neutrophils (%) | 38 ± 9.16 * | 25.3 ± 7.5 | 17.66 ± 2.08 | 34.66 ± 13.61 | 16.66 ± 4.16 | 24.66 ± 27.30 | 10–40 |
Neutrophils band (%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Immature shape (%) | 0 | 0 | 0 | 0 | 0 | 0 | 0 |