Cytotoxicity. In this study where we evaluated the cytotoxicity of crude extract and phytochemicals 1–9, and doxorubicin using RRA towards 9 carcinoma cancer cell lines; it was found that, the crude TTF extract, phytochemical 5, and doxorubicin had good cytotoxic effects against the nine (9) cancer cell lines tested. The recorded IC50 values varied from 18.32 µM (against B16-F1 murine melanoma cells) to 36.18 µM (against SKMel-505 BRAF wildtype melanoma cells) for TTF, from 10.02 µM (towards MaMel-80a BRAF-V600E homozygous mutant melanoma cells) to 31.73 µM (against SKMel-28 BRAF-V600E homozygous mutant melanoma cells) for compound 5, and from 0.22 µM (against B16-F1 cells) to 9.39 µM (against SKMel-505 cells) for doxorubicin. Eight out of 9 cell lines tested were sensitive to compound 2 with IC50 values ranging from 32.20 µM (towards B16-F1 cells) to 102 µM (against SKMel-505 cells). In the sensitive cancer cell lines, the IC50 values ranged from 28.67 µM (against MaMel-80a melanoma cells) to 109.24 µM (towards Mel-2a melanoma cells) for compound 6; and from 29.08 µM (against MaMel-80a melanoma cells) to 79.42 µM (against A2058 melanoma cells) for compound 7. It was noticed that compounds 1, 3, 4, 8, and 9 were not active above 150 µM. Rrecorded IC50 values are summarized in Table 1.
Table 1
Cytotoxicity of extracts, compounds and doxorubicin towards human melanoma and other animal cancer cell lines as determined by RRA.
Features and cell lines
|
IC50 values (µM)
|
TTF
|
2
|
5
|
6
|
7
|
Doxorubicin
|
BRAF-V600E homozygous mutant melanoma
|
MaMel-80a
|
30.18 ± 2.89
|
44.28 ± 3.29
|
10.02 ± 0.21
|
28.67 ± 3.12
|
29.08 ± 1.48
|
8.66 ± 0.56
|
SKMel-28
|
28.23 ± 1.72
|
67.28 ± 5.12
|
31.73 ± 2.20
|
33.95 ± 2.53
|
33.11 ± 2.19
|
2.14 ± 0.12
|
BRAF-V600E heterozygous mutant melanoma
|
A2058
|
19.48 ± 0.82
|
78.76 ± 5.65
|
22.77 ± 1.72
|
> 150
|
79.42 ± 4.41
|
0.29 ± 0.04
|
Mel-2a
|
18.96 ± 0.69
|
102.56 ± 8.94
|
25.45 ± 3.01
|
109.24 ± 8.44
|
> 150
|
6.63 ± 0.41
|
BRAF wildtype melanoma
|
MV3
|
28.72 ± 3.11
|
78.33 ± 6.29
|
23.52 ± 1.27
|
> 150
|
> 150
|
7.09 ± 0.59
|
SKMel-505
|
36.18 ± 2.62
|
> 150
|
22.18 ± 2.01
|
88.74 ± 6.42
|
> 150
|
9.39 ± 1.01
|
Rat colon adenocarcinoma
|
CC531
|
18.95 ± 1.27
|
54.39 ± 3.63
|
10.21 ± 0.14
|
66.38 ± 4.77
|
54.11 ± 3.10
|
0.44 ± 0.23
|
Murine melanoma
|
B16-F1
|
18.32 ± 0.96
|
32.20 ± 2.17
|
24.08 ± 0.99
|
44.90 ± 5.03
|
69.04 ± 4.95
|
0.22 ± 0.01
|
B16-F10
|
20.04 ± 1.45
|
41.85 ± 3.69
|
24.64 ± 2.56
|
50.12 ± 3.76
|
61.45 ± 5.21
|
0.24 ± 0.03
|
TTF: dichloromethane-methanol extract of TTF; 1: (3R, 4S)-3,4-dimethyloxetan-2-one; 2: luteolin; 3: stigmasterol; 4: 3-O-[6’-O-stearoyl-β-D-glucopyranosyl]stigmasterol; 5: olean-12-en-3-β-O-D-glucopyranoside; 6: 3-O-β-D-glucopyranosyl-(1→6)-β-D−glucopyranosylurs-12-en-28-oic acid; 7: 3-O-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-27-hydroxyolean-12-en-28-oic acid; 8: methyl-O-β-D-glucopyranoside; 9: β-D-fructofuranosyl-(1→4)-β-D-glucopyranoside. Compounds 1, 3, 4, 8 and 9 were not active, with no observable IC50 at up to 150 µM. In bold: significant cytotoxic effect (Boik, 2001; Brahemi et al., 2010; Kuete and Efferth, 2015); Boik, 2001; Brahemi et al., 2010) |
Acute toxicity of TTF. A single oral dose administration of 5000 mg/kg BW TTF in female rats did not cause any deaths 48 h following the administration of TTF and even during 14 days of observation. No signs of toxicity in general appearance (reduction of locomotion, stool appearance, drowsiness, salivation, reaction to noise) were observed in animals receiving this dose after 14 days of observation. Based on the OECD principle, the LD50 of the dichloromethane methanol extract of TTF was estimated to be higher than 5000 mg/kg.
Effect of TTF on food consumption and body weight. Neither death nor toxicity signs were observed on animals following 28 days of treatment with various doses of TTF (250, 500, 1000 mg/kg BW). Figures 2 and 3 summarise the data of food intake and body weight evolution respectively. No significant differences (p ≥ 0.05) in food consumption were noted in treated and untreated males, while a significant decrease (p < 0.05) in food consumption was observed in treated females until the third week when no significant differences were observed. There was no weight loss in treated male and female animals and the control in this study. However, there was a small gain in body weight in animals treated at 500 and 1000 mg/kg in males and 500 mg/kg in females compared to the control.
Effect of TTF on relative organ weight. Table 2 summarises the relative organ weight of liver, kidney, lung, heart, and spleen for male and female rats. No significant differences (p ≥ 0.05) in relative liver, kidney, lung, and heart organ weights were observed in females for all dose groups tested compared to the control group, while a significant decrease in relative spleen weight was observed for all dose levels administered compared to the control group. A significant decrease (p < 0.05) in relative liver and spleen organ weights was noted at all doses in male rats compared to the control group. However, there was no significant change in the relative weights of the kidney, lung, and heart organs in male rats.
Table 2
Effect of different doses of T. tetrapleura extract on the relative weight of organs.
Doses (mg/Kg)
|
Female
|
Male
|
0
|
250
|
500
|
1000
|
0
|
250
|
500
|
1000
|
Liver (g)
|
3.12 ± 0.09 ab
|
3.21 ± 0.07 b
|
3.10 ± 0.03 ab
|
3.09 ± 0.05 a
|
3.39 ± 0.13b
|
3.08 ± 0.02 a
|
3.10 ± 0.08 a
|
3.06 ± 0.07 a
|
Kidneys (g)
|
0.65 ± 0.02 a
|
0.69 ± 0.02 a
|
0.67 ± 0.04 a
|
0.64 ± 0.03 a
|
0.65 ± 0.03 a
|
0.66 ± 0.02 a
|
0.64 ± 0.03 a
|
0.64 ± 0.03 a
|
Lungs (g)
|
0.58 ± 0.03 ab
|
0.62 ± 0.02 b
|
0.6 5 ± 0.07 ab
|
0.56 ± 0.04 a
|
0.59 ± 0.03 a
|
0.58 ± 0.05 a
|
0.61 ± 0.02 a
|
0.65 ± 0.03 a
|
Heart (g)
|
0.30 ± 0.01 a
|
0.32 ± 0.02 a
|
0.31 ± 0.02 a
|
0.30 ± 0.02 a
|
0.32 ± 0.02 a
|
0.30 ± 0.02 a
|
0.30 ± 0.01 a
|
0.31 ± 0.02 a
|
Spleen (g)
|
0.27 ± 0.02 b
|
0.25 ± 0.02b
|
0.22 ± 0.01 a
|
0.21 ± 0.02 a
|
0.30 ± 0.06b
|
0.24 ± 0.01 b
|
0.23 ± 0.02 ab
|
0.21 ± 0.01 a
|
Data are expressed as mean ± SD, n = 4. Values in the test groups carrying the same letter as the control group in the same gender and in the same row are not significantly different according to Waller Duncan’s Multiple Comparison Test (p < 0.05) |
Effect of TTF on hematological parameters. The results of the haematological parameters (Table 3) revealed an increase (p < 0.05) in white blood cell count at 1000 mg/kg BW. In addition, there was a significant decrease (p < 0.05) in the level of HGB, HCT in female rats receiving the 1000 mg/kg dose compared to the control group. We also observed a significant decrease (p < 0.05) in PLT and PCT levels for all the different doses tested compared to the control group. In male rats, a significant decrease (p < 0.05) was observed at 1000 mg/kg for HCT and PCT levels, and at all doses tested for PLT levels. There was also a significant increase in GR and a non-significant increase in WBC compared to control.
Table 3
Hematological parameters in male and female rats after 28 days of administration
Sexe
|
Female
|
Male
|
Dose (mg/kg)
|
0
|
250
|
500
|
1000
|
0
|
250
|
500
|
1000
|
WBC (103/µL)
|
4.60 ± 0.36ab
|
4.87 ± 0.12a
|
5.30 ± 0.61bc
|
5.80 ± 0.35c
|
5.47 ± 0.31a
|
5.67 ± 0.78a
|
6.10 ± 0.66a
|
6.00 ± 0.60a
|
LY (%)
|
65.87 ± 1.76ab
|
64.03 ± 1.80a
|
65.50 ± 3.11ab
|
69.30 ± 1.91b
|
65.80 ± 4.39a
|
62.93 ± 2.69 a
|
62.80 ± 5.30 a
|
62.37 ± 3.32 a
|
MO (%)
|
6.12 ± 0.55a
|
5.13 ± 0.61a
|
5 ± 0.89a
|
5.83 ± 0.70a
|
4.37 ± 0.81ab
|
5.70 ± 0.96b
|
6.35 ± 1.2b
|
3.73 ± 0.95a
|
GR (%)
|
27.80 ± 1.77ab
|
30.83 ± 1.65b
|
30.50 ± 2.69b
|
25.97 ± 0.76a
|
26.80 ± 0.30a
|
30.97 ± 1.72b
|
32.60 ± 2.78b
|
32.20 ± 1.51b
|
RBC (106/µL)
|
8.56 ± 0.48a
|
8.66 ± 0.46a
|
8.69 ± 0.67a
|
8.25 ± 0.21a
|
9.12 ± 0.48a
|
8.70 ± 0.31a
|
8.50 ± 0.18a
|
8.94 ± 0.45a
|
HGB (g/dL)
|
18.30 ± 0.66b
|
18.27 ± 0.76b
|
17.70 ± 0.72b
|
16.20 ± 0.46a
|
17.53 ± 0.12a
|
17.5 ± 0.93a
|
16.50 ± 0.26a
|
17.33 ± 0.90a
|
HCT (%)
|
54.43 ± 1.66b
|
54.13 ± 1.56b
|
51.97 ± 2.22ab
|
47.77 ± 2.92a
|
54.57 ± 2.94b
|
51.03 ± 1.83ab
|
52.30 ± 2.46ab
|
49.47 ± 2.02a
|
MCV (fL)
|
60.10 ± 0.78b
|
60.43 ± 1.19b
|
59.67 ± 1.07ab
|
57.90 ± 0.79a
|
60.50 ± 1.15 a
|
59.10 ± 1.20a
|
59.53 ± 0.65 a
|
60.17 ± 0.80 a
|
MCH (pg)
|
19.87 ± 0.31a
|
20.30 ± 0.72a
|
19.90 ± 0.61a
|
19.83 ± 0.35a
|
18.70 ± 0.52 a
|
19.53 ± 0.75a
|
19.20 ± 0.53a
|
19.92 ± 0.78a
|
MCHC (g/dL)
|
33.63 ± 1.14a
|
33.10 ± 0.50a
|
34.43 ± 0.85a
|
33.53 ± 0.45a
|
31.63 ± 1.12 a
|
32.53 ± 1.07 a
|
31.90 ± 0.70 a
|
33.70 ± 1.00 a
|
RDWCV (%)
|
15.87 ± 0.21a
|
16.97 ± 1.53a
|
16.60 ± 0.61a
|
16.83 ± 0.93a
|
16.47 ± 0.32a
|
16.53 ± 0.15a
|
15.90 ± 0.56a
|
17.23 ± 1.50a
|
RDWSCD (fL)
|
36.53 ± 1.64a
|
41.3 ± 2.18b
|
38.43 ± 0.55a
|
41.17 ± 1.50b
|
40.10 ± 0.36 a
|
39.50 ± 1.37 a
|
37.87 ± 1.76 a
|
40.73 ± 4.62 a
|
PLT (103/µL)
|
888.67 ± 21.46b
|
730.33 ± 32.15a
|
717.33 ± 25.74a
|
727.33 ± 44.77a
|
666.33 ± 29.16b
|
609 ± 41.8ab
|
597.67 ± 24.38a
|
569.67 ± 19.14a
|
PCT (%)
|
0.61 ± 0.02b
|
0.46 ± 0.06a
|
0.49 ± 0.02a
|
0.50 ± 0.03a
|
0.49 ± 0.03b
|
0.43 ± 0.05ab
|
0.46 ± 0.02b
|
0.37 ± 0.02a
|
PDW (fL)
|
17.57 ± 0.23a
|
17.33 ± 0.55a
|
17.00 ± 0.53a
|
17.10 ± 0.75a
|
17.03 ± 0.47ab
|
18 ± 0.50b
|
16.73 ± 0.29a
|
17.63 ± 0.29b
|
MPV (fL)
|
6.77 ± 0.12a
|
7.02 ± 0.27a
|
6.83 ± 0.12a
|
6.83 ± 0.12a
|
6.90 ± 00 a
|
6.97 ± 0.31 a
|
6.60 ± 0.17 a
|
6.90 ± 0.20 a
|
Data are expressed as mean ± SD, n = 4. Values in the test groups carrying the same letter as the control group in the same-sex and in the same row are not significantly different according to Waller Duncan’s Multiple Comparison Test (p < 0.05). WBCs: White blood cells, RBCs: Red blood cells, HCT: Hematocrit, PLT: Platelets, HGB: Haemoglobin, MCH: Mean corpuscular haemoglobin, MCHC: Mean corpuscular haemoglobin concentration, MCV: Mean corpuscular volume, Gran: Granulocytes, Lym: Lymphocytes, MO: Monocytes, RDWCV: Red blood cells distribution width CV, RDWSD: Red blood cells distribution width SD, PCT: Plateletcrit, MPV: mean platelet volume, PDW: Platelet distribution width. |
Effect of TTF on urinary biochemical parameters. The results of the urinary and serum biochemical parameters of rats treated with methanol extract of TTF are presented in Table 4. The extract did not affect urinary protein levels in either sex. There was a significant decrease (p < 0.05) in urinary creatinine from the 500 mg/kg dose onwards in female rats whereas no significant difference was observed in males compared to the control group. In both sexes, there was a significant increase (p < 0.05) in urine urea at 1000 mg/kg in females and 500 mg/kg in males compared to the control groups.
Table 4
Urinary biochemical parameters in males and females after 28 days of administration
Sex
Doses (mg/Kg)
|
Female
|
Male
|
0
|
250
|
500
|
1000
|
0
|
250
|
500
|
1000
|
Creatinine (mg/dL)
|
2.85 ± 0.13b
|
2.62 ± 0.16b
|
2.04 ± 0.06a
|
2.04 ± 0.06a
|
4.11 ± 0.04b
|
3.86 ± 0.05a
|
3.68 ± 0.04a
|
3.49 ± 0.06a
|
Urea (mg/dL)
|
107.23 ± 4.26a
|
104.07 ± 3.66a
|
108.43 ± 2.25a
|
130.12 ± 2.51b
|
106.93 ± 2.67a
|
113.41 ± 4.03a
|
131.93 ± 4.69b
|
133.73 ± 1.77b
|
Total Protein (g/dL)
|
0.79 ± 0.05a
|
0.79 ± 0.05a
|
0.85 ± 0.05a
|
0.83 ± 0.06a
|
0.82 ± 0.03a
|
0.86 ± 0.06a
|
0.85 ± 0.05a
|
0.80 ± 0.06a
|
Data are expressed as mean ± SD, n = 4. Values in the test groups carrying the same letter as the control group in the same gender and in the same row are not significantly different according to Waller Duncan’s Multiple Comparison Test (p < 0.05) |
Effect of TTF on serum biochemical parameters. Table 5 shows the results of serum biochemical parameters of the animals in the control groups and those that received different TTF doses (250, 500, and 1000 mg/kg BW). In both male and female rats, after prolonged administration, a significant decrease (p < 0.05) in the values of transaminase activity (ALAT and ASAT), alkaline phosphatase, and total protein concentration were observed for all doses administered in comparison with the control group. In addition, a significant increase (p < 0.05) in urea levels was observed in male rats at all doses tested but was only observed in female rats at doses above 1000 mg/kg. A significant decrease in serum creatinine was observed in male rats at all doses tested compared to the control group (p < 0.05).
Table 5
Serum biochemical parameters in males and females after 28 days of administration
Doses (mg/Kg)
|
Female
|
Male
|
0
|
250
|
500
|
1000
|
0
|
250
|
500
|
1000
|
Creatinine (mg/dL)
|
0.78 ± 0.02a
|
0.78 ± 0.05a
|
0.85 ± 0.05a
|
0.84 ± 0.03a
|
1.44 ± 0.03c
|
1.33 ± 0.02b
|
1.27 ± 0.01a
|
1.28 ± 0.02a
|
Urea (mg/dL)
|
71.23 ± 3.35a
|
71.23 ± 1.58a
|
73.80 ± 7.27a
|
87.35 ± 2.56b
|
62.35 ± 0.78a
|
70.48 ± 0.98b
|
72.29 ± 2.25bc
|
74.25 ± 1.86c
|
ASAT (U/L)
|
107.18 ± 2.31b
|
106.96 ± 1.49b
|
94.62 ± 2.57a
|
97.56 ± 1.82a
|
105.43 ± 2.31c
|
84.43 ± 3.23b
|
73.71 ± 2.61a
|
73.06 ± 1.12a
|
ALAT (U/L)
|
61.19 ± 1.82b
|
57.31 ± 1.13a
|
57.75 ± 1.43a
|
57.09 ± 1.31a
|
65.19 ± 2.08c
|
57.53 ± 1.66b
|
53.81 ± 2.53a
|
54.03 ± 1.49a
|
PAL (U/L)
|
363.43 ± 3.12d
|
308.26 ± 1.49c
|
261.29 ± 3.12b
|
200.18 ± 1.75a
|
420.43 ± 2.35c
|
376.66 ± 2.35b
|
373.46 ± 3.12b
|
352.94 ± 2.35a
|
Total Protein (g/dL)
|
8.70 ± 0.29b
|
7.66 ± 0.22a
|
7.37 ± 0.58a
|
7.02 ± 0.42a
|
10.31 ± 0.07b
|
9.13 ± 0.85a
|
8.38 ± 0.70a
|
7.92 ± 0.59a
|
Data are expressed as mean ± SD, n = 4. Values in the test groups carrying the same letter as the control group in the same gender and in the same row are not significantly different according to Waller Duncan’s Multiple Comparison Test (p < 0.05). ASAT: Aspartate amino transferase, ALAT: Alanine amino transferase, PAL: Phosphatase alkaline |
Effect of TTF on lipid profile of male and female rats after 28 days of oral treatment. All the lipid profile values obtained from the serum of the control and test groups at different doses (250, 500, and 1000 mg/kg) after 28 days of oral administration are presented in Table 6. A significant decrease (p < 0.05) in triacylglycerol (TAG) was observed in female rats from the dose of 500 mg/kg. LDL cholesterol varied significantly, by decreasing (p < 0.05) at all doses tested in both males and females compared to the control group. A significant increase (p < 0.05) in total cholesterol and HDL cholesterol values was observed in both males and females compared to the control group for all different doses tested.
Table 6
Lipid profile parameters in females and males after 28 days of administration
Doses (mg/Kg)
|
Female
|
Male
|
0
|
250
|
500
|
1000
|
0
|
250
|
500
|
1000
|
TAG (mg/kg)
|
62.45 ± 2.25b
|
62.83 ± 1.29b
|
57.23 ± 3.09a
|
50.94 ± 3.96a
|
67.17 ± 3.08a
|
71.13 ± 3.28a
|
69.81 ± 2.35a
|
65.85 ± 3.04a
|
T-CHOL (mg/Kg)
|
82.60 ± 1.77 a
|
90.54 ± 1.57 b
|
95.96 ± 2.89c
|
103.78 ± 3.2d
|
88.78 ± 1.30a
|
93.82 ± 3.80ab
|
97.23 ± 4.59b
|
112.86 ± 2.58c
|
HDL (mg/Kg)
|
52.71 ± 2.12 a
|
62.04 ± 0.71b
|
68.98 ± 3.01c
|
81.34 ± 3.17d
|
53.68 ± 1.94a
|
66.33 ± 2.78b
|
69.99 ± 4.84b
|
83.23 ± 2.97c
|
LDL (mg/Kg)
|
18.70 ± 1.14c
|
15.93 ± 1.30b
|
15.51 ± 1.38b
|
12.26 ± 1.03a
|
21.66 ± 0.84b
|
13.26 ± 2.27a
|
13.28 ± 1.28a
|
16.46 ± 2.78a
|
Data are expressed as mean ± SD, n = 4. Values in the test groups carrying the same letter as the control group in the same-sex and in the same row are not significantly different according to Waller Duncan’s Multiple Comparison Test (p < 0.05). TAG: Triacylglycerol, T-CHOL: Total cholesterol, HDL: High density lipoprorotein, LDL: Low density lipoprotein |
Histological sections of the liver and kidneys. Histopathological examinations were performed on the liver and the kidney to assess organ damage. The kidney of treated rats showed normal glomeruli and there was no necrosis of tubular epithelium either in female (Fig. 4) or male (Fig. 5) treated rats. Adverse effects were neither found in the liver of female (Fig. 6) nor male (Fig. 7) treated rats compared to the control group.