Effect of Moringa oleifera Lam. Seed Extract on Toluene Diisocyanate-Induced Immune-Mediated Inflammatory Responses in Rats

Figures & data

TABLE 1 Preliminary phytochemical study on M.oleifera seed extract

Tests	Observation
Alkaloids
Wagner's test	Positive—Turbidity noted
Mayer's test	Positive—Turbidity noted
Flavonoids
Dilute HCL	Positive—Precipitation noted
Glycosides
Bromine test	Positive—Turbidity noted
Cupraloin test	Positive—Turbidity noted
Saponins
Sodium bicarbonate	Negative—No change observed
Polyphenols
Ferric chloride test	Negative—No change observed
Lead acetate test	Negative—No change observed
Steroids
Liebermann-Burchard's test	Negative—No change observed
Salkowski test	Negative—No change observed
Tannins
Ferric chloride test	Positive—Opacity noted
Lead acetate test	Positive—Opacity noted
Triterpenoids
Acetic anhydride test	Positive—Precipitation noted

TABLE 2 List of major phytochemical constituents of the seeds of M. oleifera

Active principle/s (isolated/reported from seed extract)	Reported activity	Reference
Alkaloid, Moringine	Relaxes bronchioles	Kirtikar and Basu, 1975
Flavonoids, Tocopherols, vitamin C and different polyphenolic compounds	Antioxidant	Laandrault et al., 2001
Glycosides, Benzylisothiocyanate, its derivatives (a glycoside of mustard oil) 4 (4′-acetyl-L-L-rhamnosyloxy)- benzoisothiocyanate	Anti-inflammatory External counter- irritant property	Das et al., 1958
4 [α -L-rhamnosyloxy], Phenylacetonitrile, 4-hydroxyl phenylacetonitrile and 4-hydroxyphenylacetamide	As mutagens	Villasenor et al., 1989, 1990; Villasenor, 1994
4-[α -L-rhamnosyloxy]	Antibiotic	Dayrit et al., 1990
4-[[α -L-rhamnosyloxy] phenyl acetonotrile β -Carotene], β -sterols, and lecithin	Antimicrobial	Njoku and Adikwu, 1997
O-ethyl-4-(α -L-rhamnosyloxy)benzylcarbamate together with seven known compounds like-niazimicin, niazirin, β -sitostrol, glycerol-1-1-(9-octadecanoate), 3-O-(6′-O-oleoyl-β -D-glucopyranosyl)-β -sitosterol, and β -sitosterol-3-O-β -D-glucopyranoside	Antitumor promoting activity	Guevara et al., 1999
Thiamine, riboflavin, nicotinic acid, folic acid, pyridoxine, ascorbic acid and α -tocopherol	As nutritive and as an antioxidant	Dahot and Memon, 1985
Monopalmitic acid, oleic acid, and tri-oleic triglycerides were isolated from seeds and oil	−	Memon and Khatri, 1987

TABLE 3 Total cells and differential leukocyte counts in blood (×10³ cells/ml)^a

Groups	Total cells	Eosinophils	Lymphocytes	Monocytes	Neutrophils
Control	15.00 ± 0.87	0.06 ± 0.01	13.2 ± 0.80	0.040 ± 0.007	1.7 ± 0.06
TDI-control	16.48 ± 1.02	0.45 ± 0.004^c	13.5 ± 0.74	0.039 ± 0.004	2.5 ± 0.24^b
TDI + DEXA (2.5 mg/kg)	14.24 ± 0.73	0.08 ± 0.01^c	13.08 ± 0.57	0.038 ± 0.007	1.75 ± 0.15^b
TDI + MO1 (100 mg/kg)	16.25 ± 0.84	0.12 ± 0.027^c	13.6 ± 0.25	0.042 ± 0.007	1.79 ± 0.16^b
TDI + MO2 (200 mg/kg)	14.97 ± 0.75	0.09 ± 0.011^c	13.1 ± 0.67	0.042 ± 0.006	1.74 ± 0.07^b

^aValues are expressed as mean ± SEM (n = 8/regimen). Comparisons were made between Group-I (non-sensitized control) and Group-II (sensitized control), and between Group-II and Groups-III (dexamethasone-treated), IV (MO1-treated), and V (MO2-treated).

^b p < 0.01;

^c p < 0.001.

TABLE 4 Total cells and differential leukocyte count in BAL fluid (×10⁵ cells/ml)

Groups	Total cells	Eosinophils	Lymphocytes	Macrophages	Neutrophils
Control	13.85 ± 0.48	0.05 ± 0.009	1.4 ± 0.13	12.3 ± 0.34	0.08 ± 0.017
TDI-control	47.95 ± 1.96^c	1.54 ± 0.14^c	4.17 ± 0.31^c	42.17 ± 1.45^c	0.87 ± 0.06^c
TDI+DEXA (2.5 mg/kg)	17.34 ± 1.37^c	0.26 ± 0.13^c	1.33 ± 0.14^c	15.52 ± 1.0^c	0.23 ± 0.10^b
TDI+MO1 (100 mg/kg)	20.82 ± 1.94^c	0.84 ± 0.19^a	3.07 ± 0.24^a	16.58 ± 1.3^c	0.43 ± 0.21
TDI+MO2 (200 mg/kg)	19.24 ± 1.69^c	0.52 ± 0.21^c	2.5 ± 0.36^b	16.01 ± 1.1^c	0.21 ± 0.02^b

Values are expressed as mean ± SEM (n = 8/regimen). Comparisons were made between Group-I (non-sensitized control) and Group-II (sensitized control), and between Group-II and Groups-III (dexamethasone-treated), IV (MO1-treated), and V (MO2-treated).

^a p < 0.05;

^b p < 0.01;

^c p < 0.001.

FIG. 1 Serum TNFα levels in rats. *Value significantly different from non-arthritic control (p < 0.001); value different from TDI-controls (^$ p < 0.05). Values shown are the mean ± SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

FIG. 2 Serum IL-4 levels in rats. *Value is significantly different from nonsensitized control (p < 0.001); value significantly different from TDI-controls (^@ p < 0.001, ^# p < .01). Values shown are the mean ± SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

FIG. 3 Serum IL-6 level in rats. *Value is significantly different from non-sensitized control (p < 0.001); value significantly different from TDI-controls (^# p < 0.01, ^$ p < 0.05). Values shown are the mean ±SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

FIG. 4 BAL fluid TNFα levels in rats. *Value is significantly different from nonsensitized control (p < 0.001); value significantly different from TDI-controls (^# p < 0.01, ^$ p < 0.05). Values shown are the mean ± SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

FIG. 5 BAL fluid IL-4 levels in rats. *Value is significantly different from control (p < 0.001); value significantly different from TDI-controls (^@ p < 0.001). Values shown are the mean ± SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

FIG. 6 BAL fluid IL-6 levels in rats. *Value is significantly different from control (p < 0.001); value significantly different from TDI-controls (^@ p < 0.001, ^# p < 0.01, ^$ p < 0.05). Values shown are the mean ± SEM from non-sensitized controls, sensitized controls (DC), and treatment regimen rats (DEXA = dexamethasone; MO1 = MOEE 100 mg/kg; MO2 = MOEE 200 mg/kg; n = 8 rats/group).

TABLE 5 Effect of MOEE on oxidative stress in TDI sensitized rats

	Antioxidant enzymes
Treatment Groups	SOD (U/mg protein)	CAT (μ mole H2O2 consumed/min/mg protein)	GSH (μ g/mg protein)	Pro-oxidant MDA (μ mole/mg protein)
Control (Distilled water)	3.57 ± 0.11	85.57 ± 2.4	9.53 ± 0.25	0.16 ± 0.04
TDI-control	3.26 ± 0.07^c	35.83 ± 1.2^c	4.44 ± 0.4^c	0.31 ± 0.05^c
TDI+DEXA (2.5 mg/kg)	3.61 ± 0.06^c	61.57 ± 2.5^c	10.7 ± 0.33^c	0.18 ± 0.08^b
TDI+MO1 (100 mg/kg)	3.33 ± 0.12	35.56 ± 1.5	8.93 ± 0.38^c	0.31 ± 0.029
TDI+MO2 (200 mg/kg)	3.58 ± 0.09^c	43.64 ± 1.6^a	9.13 ± 0.31^c	0.23 ± 0.018^a

Values are expressed as mean ± SEM (n = 8/regimen). Comparisons were made between Group-I (non-sensitized control) and Group-II (sensitized control), and between Group-II and Groups-III (dexamethasone-treated), IV (MO1-treated), and V (MO2-treated).

^a p < 0.05;

^b p < 0.01;

^c p < 0.001.

FIG. 7 Histopathological effects from the various treatments. Lung tissue sections of treated and untreated TDI sensitized animals stained with hematoxylin-eosin. (A) Shows normal view of lung pathology. (B) Shows typical damaged lung tissue from TDI-control group with total and differential leukocyte infiltration (eosinophils), reduced lumen size. (C) Shows less leukocyte infiltration lumen size by dexamethasone treatment. (D) MO1 shows less leukocyte infiltration but does not protected animal from other pathological features. (E) MO2 shows significant protection against leukocyte infiltration, and constriction of lumen size.

Kirtikar K. R., Basu B. D. Indian Medicinal Plants, 2nd Edition, D. Dun, B. Singh, M. P. Singh. Bishen Singh Mahendra Pal Singh, New Cannaught Place-Dehradun 1975; Vol. 1: 676–683

 Google Scholar

Laandrault N., Pouchert P., Ravel P., Gase F., Cros G., Teissedro P. L. Antioxidant activities phenolic level of French wines from different varieties and vintages. J. Agric. Food Chem. 2001; 49: 3341–3343

 PubMedGoogle Scholar

Das B. R., Kurup P. A., Narasimha Rao P. L. Antibiotic principle from Moringa Pterosperma Part IX. Inhibition of transaminase by isocyanates. Ind. J. Med. Res. 1958; 46: 75–77

 PubMedGoogle Scholar

Villasenor I. M., Lim-Sylianco C. Y., Dayrit F. Mutagens from roasted seeds of Moringa oleifera. Mutat. Res. 1989; 224: 209–212

 PubMed Web of Science ®Google Scholar

Villasenor I. M., Dayrit F., Lim-Sylianco C. Y. Studies on M. oleifera seeds, Part II. Thermal degradation of roasted seeds. Phil. J. Sci. 1990; 119: 33–39

 Google Scholar

Villasenor I. M. Bioactive metabolites from Moringa oleifera Lam. Kimika 1994; 110: 47–52

 Google Scholar

Dayrit F. M., Alcantara A. D., Villasenor I. M. The antibiotic compound and its deactivation in aqueous solution. Phil. J. Sci. 1990; 119: 23–26

 Google Scholar

Njoku O. U., Adikwu M. U. Investigation on some physicochemical antioxidant and toxicological properties of Moringa oleifera seed oil. Acta. Pharma. Zagr. 1997; 47: 287–290

 Google Scholar

Guevara A. P., Vargas C., Sakurai H., Fujiwara Y., Hashimoto K., Maoka T., Kozuka M., Ito Y., Tokuda H., Nishino H. An antitumor promoter from Moringa oleifera Lam. Mutat. Res. 1999; 440: 181–188

 PubMed Web of Science ®Google Scholar

Dahot M. U., Memon A. R. Nutritive significance of oil extracted from Moringa oleifera seeds. J. Pharm. Univ. Karachi 1985; 3: 75–80

 Google Scholar

Memon G. M., Khatri L. M. Isolation and spectroscopic studies of mono-palmitic, di-oleic triglyceride from seeds of Moringa oleifera Lam. Pak. J. Sci. Ind. Res. 1987; 30: 393–395

 Google Scholar