3.1. Phytochemical study.
3.1.1. Isolation and Identification of metabolites.
The crude leaf extract of MA (70% hydroalcoholic) was applied to polyamide 6 column chromatography and eluted with water methanol mixtures in the order of decreasing polarity.
All collected fraction was investigated individually by TLC chromatography.
The 20-50 % methanolic/water subfractions uploaded on polyamide 6 column using water/methanol in order of decreasing polarity. The collected subfractions was purified by Sephadex LH-20 using 50% ethanol /water as eluent to yield 5 compounds identified as, quercetin, quercetin-3-O-β-D-glucoside, luteolin-7-O-β-D-glucopyranoside and Gallic acid, cinnamic acid. On the other hand, the 70 % alcohol subfraction was applied to Sephadex LH-20 column chromatography and eluted with 80% ethanol/water to give one pure compound as 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one(fig1).
These compound were identified by comparing their spectral data with those reported.
Compound 1 was identified as trans-cinnamic acid; it is soluble in chloroform and methanol. Moreover, Compound 1 gives blue color under UV light at 254 nm with RF: 0.87, 1H NMR (CD3OD, 400 MHz): δ ppm = 7.63(d, J= 15.9, 1H, H-β), the remaining phenyl ring resonate at δppm 7.61 –7.57 (m, 2H) and 7.42 – 7.38 (m, 3H), 6.51 (d, J = 15.9, 1H, H-α) and 13C NMR (CD3OD, 400 MHz): δ ppm :171.65 (C=O), 145.05 (C-β), 136.16(C-1), 131.05(C-4), 129.93 (C2 & C-6), 129.00 (C-3 & C-5), 120.99 (C-α) in (Fig S2 and Table 3) .
Compound 2 identified as gallic acid: It soluble in methanol and gave light blue color under UV at 254 nm. 1H NMR analysis (400 MHz, DMSO-d6) δ ppm: 6.97 (S). 13C NMR: (δ ppm) 165.67 (C7; C=O) 144.10 (C3& C5), 138.99 (C4), 118.82 (C1) 108.86 (C2& C6) (Fig S3 and Table 3) .
Compound 3 was identified as quercetin; It is soluble in methanol and give yellow color under UV light, 356 nm, 1H NMR (400 MHz, DMSO): δ 7.74 (1H, d, J = 2.1 Hz, H-2’), 7.62 (1H, dd, J = 8.3, 2.1 Hz, H-6’), 6.88 (1H, d, J = 8.3 Hz, H-5’), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-6)” and 13C NMR: δ (ppm) 93.2 (C-8), 98.0 (C-6), 102.9 (C-10), 115.0 (C-2'), 115.4 (C-5'), 119.8 (C-6'), 121.8 (C-1'), 135.5 (C-3), 144.9 (C3'), 146.7 (C-2), 147.5 (C-4'), 156.0 (C-9), 160.6 (C-5), 163.8 (C-7), 176.7 (C-4). Compounds 1,2 and 3 were eluted with mobile phase benzene: methanol: acetic acid,45:8:4 (Fig S4 and Table 3) .
Compound 4 identified as as quercetin-3-O-β- D-glucoside; it soluble in methanol gave dark purple color under UV light at 254 nm with RF : 0.42 eluted with mobile phase butanol: acetic acid: water, 30:10:10. 1H NMR (400 MHz, DMSO-d6: 7.59 (1H, d, J = 1.8, H-2), 7.62 (1H, dd, J = 1.8 & 8.4, H-6’), 6.87 (1H, dd, J =8.4, H-5’), 6.39 (1H, d, J = 2.2, H-8), 6.19 (1H, d, J = 2.4, Í-6), 5.43 (1H, d, J = 7.6,H-1’’13C NMR spectrum (100 MHz, DMSO-d6, , ppm): 156.39 (C-2), 133.31 (C-3), 178.18 (C-4), 161.12 (C-5), 98.66 (C-6), 163.23 (C-7), 94.13 (C-8), 156.14 (C-9), 104.75 (C-10), 120.88 (C-1‘), 117.04(C-2’), 145.25 (C-3’), 148.78 (C-4’), 114.28 (C-5’), 122.01 (C-6’), 101.09 (C-1’’), 74.16 (C-2’’), 76.73 (C-3’’), 70.89 (C-4”), 77.65 (C-5’’), 61.18 (C-6’’) (Fig S5 and Table 3) .
Compound 5 was identified as Luteolin-7-O-β-D-glucopyranoside, It soluble in methanol and give dark purple under UV lamb at 254 nm with RF: 0.891 eluted by mobile phase butanol: acetic acid: water, 30:10:10. 1H-NMR (DMSO-d6) revealed signals at d ppm 7.45 (dd, J = 8.3 Hz, and J = 2.2 Hz, H-6'), 7.42 (d, J = 2.2 Hz, H-2'), 6.9 (d, J = 8.3 Hz, H-5'), 6.8 (d, J = 2.2 Hz, H-8), 6.7 (s, H-3), 6.4 (d, J = 2.2 Hz, H-6) and signal appeared as doublet at d ppm 5.08 (d, J = 6.6 Hz, H-1'' of glucose) assignable for the anomeric proton of the sugar moiety and 13C-NMR spectrum (DMSO-d6). 13C NMR (100MHZ, DMSO-d6) δ (ppm): 181.92 (C-4), 164.99 ( C-7), 163.14 (C-2), 161.51 (C-5), 157.97 (C-9), 149.95 (C-4′), 147.12 (C-3′), 118.88 (C-6′), 122.12 (C-1′), 116.59 (C-5′), 113.86 (C-2′), 103.89 ( C-10), 103.19 (C-3), 99.82 (C-1′′), 99.58 (C-6), 94.49 (C-8), 77.14 ( C-5′′), 76.71 (C-3′′), 74.02 (C-2′′), 70.11 (C-4′′), 61.17 (C-6′′) (Fig S6 and Table 3) .
Compound 6 was identified as 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one, it soluble in chloroform and gave light blue under UV lamb, 254 nm. with RF : 0.82 eluted by mobile phase methanol: chloroform, 9.5:0.5.1H NMR (400 MHz, CDCl3), δppm : 392(3H s, OCH3,)7.05 (2H, d, J=8.48 Hz, H-3′ and H-5′), 7.34(1H, s, H-3), 7.42 (2H, d, J= 8.48, H-2’ and H-6’),7.58 (1H, d, J= 8.49, H-5), 7,81(1H, t, J=8.49 Hz, H-8), 7.95 (1H, d, J=8.49, H-6), 8.27 (1H, dd, J =1.2 & 8.3Hz, H-7), 8.78 (1H, dd, J=1.2 &8.3, H-9); 13CNMR(CDCI3 ppm: 55.60 (OCH3), 112.89 (C-3), 114.15 (C-3’ and C-5’), 125.04 (C-9b), 126.63(C-3a), 127.77(C-8), 129.84 (C-9a), 130.14 (C-5), 131.38 (C-6), 131.65(C-6a), 131.72(C-9). 132.00 (C-2’ and C-6’), 132.39 (C-1’), 136.64 (C-7), 143.99 (C-4), 149.49 (C-2), 159.75(C-4’), 179.98 (C-l) (Fig S4 and Table 3) .
3.1.2. GC-MS of essential oil from fruit peels.
The essential oil was extracted from fruit peels by hydrodistillation from five fours which is the suitable time to obtain the volatile oils.
Injection obtained essential oil to GC/MS resolute 37 compounds. The major compounds was isoamyl isobutyrate amounted to 18.3%, followed by Myristicine 9.31% and Isovaleric acid amounted to 8.06% of the oil. On the other hand, the oil contained n-Hexadecanoic acid amounted to 22.05%. All compounds identified are uncommon compounds found in essential oil as general. However these compounds are similar to that identified from banana reported by Heliofabia et al., . These constituents of fruit peel were presented in (Table 2).
HPLC and LC-MSMS profiles of secondary metabolites from MA.
The metabolomics profile was identified based on low and high throughput sensitive LC/MS analyses which enabled the in-depth studies of secondary metabolite changes in MA plant with different parts as leaves, pesudostem and in fruit peels. 76 different compounds were identified including phenols, flavonoids, phenylphenalenones, amino acids and fatty acids from the agro waste of different parts of MA. LC-MSMS profile was used as a marker for the Ulcerative colitis. The individual compounds were identified via comparison of the exact molecular masses (∆ less than 5 ppm, mass spectra and retention times) with those of the standard compounds available in PubChem, ChEBI, Metlin, KNApSAck, HPLC, NMR and literature data. Different types of phenolic compounds of MA extracts were recorded in (Table 3) included phenolic acids and polyphenols such as gallic acid, caffeic acid, syringic acid, ferulic acid, Salicylic acid, Caffeic acid, Caffeoylquinic acid, kaempferol, catechin, Feruloylquinic acid, Vanillic acid hexoside, Sinapic acid-O-glucoside, and Kaempferol 3-Sophortrioside.
The biologically effects of Musa extract are most probably due to its content found in the different extracts. These fractions included the petroleum ether, chloroform fraction, ethyl acetate fraction, n-butanol fraction, and water fraction. In the biological activity screening tests, the n-butanol fraction showed stronger antioxidant activities than the other four fractions and it was also the potent fraction for in vivo efficacy study of the protective effects against ulcerative colitis.
Metabolomics based on high throughput sensitive UPLC-HESI-MSMS enabled in-depth studies on secondary metabolites in several parts from MA and revealed 75 different compounds, mainly phenolic, flavonoids and 12 different fatty acids. The individual compounds were identified via the exact molecular masses with ∆ less than 5 ppm, mass spectra and retention times and were compared with those of the standard compounds, as well as databases available online (PubChem, ChEBI, Metlin and KNApSAck) and literature data (Table 3).
Excessive production of cytokines as Ilβ6 lead to severe inflammation which can be suppressed by natural compounds as phenolics present in natural products like p-coumaric acid , rutin caffeic acid which inhibits induction of lipopolysaccharide inducible nitric oxide synthase production , also flavonoids as naringenin, quercetin prevent expression of inducible nitric oxide synthase protein through inhibition of nuclear factor-κB that represents the major transcripting factor for inducible nitric oxide synthase .
In the current work, a comprehensive characterization of secondary metabolites using LC/MSMS was accomplished in the hydroalcoholic Musa waste extract, as well as in the oil fraction identified by GC/MS. The analysis explained 75 secondary metabolites belonging to simple phenols, amino acids, phenolic acids, cinnamic acid derivatives and flavonoids in addition to sugars. Total flavonoid and phenolic contents were more pronounced in the butanol extract. The latter also exhibited potent anti-inflammatory bowel disease “Ulcerative Colitis”
Phenolic acids are aromatic carboxylic acid with hydroxyl derivatives that have only one phenolic ring in their structure. They include two types; hydroxybenzoic acid and hydroxycinnamic acid derivatives . Caffeic, p-coumaric, ferulic and sinapic acids are the hydroxycinnamic acid derivatives that are more abundant in plants as compared to the benzoic acid derivatives; such as gallic acid, protocatechuic acid and p-hydroxybenzoic acid (Table 3).
3.2. Pharmacological study.
3.2.1. In vitro study
Results of the present study revealed that the highest concentrations of IC50 in both DPPH and ABTS antioxidant were found in MeOH-Leaves; 5.85: 14.92, then MeOH-fruit; 9.94:12.08 and MeOH- pesudostem; 13.17:41.08, respectively) Fig 2 and Table S2. These findings are in agreement with Oresanya et al. .
3.2.2. Acute and sub chronic toxicity studies:
In the present acute toxicity study Musa leaves, pseudo-stem and fruits extracts given to three groups rats in a single dose of 5 gm/kg; all were given once; exhibited no mortalities during the first twenty four hours after administration. The percentage of body weight change of the group that received pseudo-stem extract showed significant decrease while the group that received fruit extract showed significant increase compared to negative control group (Table 4). However there weren’t any changes in bowel habits, also there weren’t any changes in behaviour or hair loss or discolouration in all groups during the two successive weeks duration of the experiment.
Moreover histopathologic examination of both liver and kidneys revealed normal hepatic parenchyma and normal hepatocytes (fig 5a), and normal renal tubules and glomeruli (fig 6a).
Accordingly the selected doses for testing the sub-chronic toxicity of all extracts were 250 and 500 mg/kg given orally for fourteen successive days, which is the same duration of the efficacy study. Observation of rats for any marked change in body weights (Table 5), or gross bowel habit changes as severe or frequent motions or severe constipation revealed that they were the same as negative control group, also their behaviour was the same as negative control group.
Assessment of both liver and kidney functions in the subchronic toxicity study by measuring ALT, AST, Urea and Creatinine levels in sera of treated rats (Table 6), in the subchronic toxicity study showed non - significant variation from negative control group.
In the present study, results of both acute and subchronic toxicity studies denoted the safety of Musa leaves, pseudostem and fruits to be used in the efficacy study as protective agents against inflammatory model of rat distal part of colon mimicking ulcerative colitis in humans.
3.2.3. Efficacy Study:
In the present study the efficacy of Musa leaves, pseudo-stem and fruit extracts was evaluated as potential protective supplements against colonic inflammatory disease in a rat model mimicking ulcerative colitis in human patients.
Ulcerative colitis was induced by per rectal injection of 2ml 8% acetic acid. Treatment with Musa extracts was given orally to rats in doses of 250 and 500 mg/kg of each extract for fourteen days prior to induction of ulcerative colitis. The doses were selected according to the results of toxicity studies formerly done in the present work.
The weights of all treated rats involved in the study were within normal and didn’t show any significant difference from the negative control group, also the % change of weights at the end of experiment compared to those before starting was minimal. The non significant change in body weights denotes that the extracts don’t alter normal bowel habits and don’t affect the appetite of rats as food consumption was constant throughout the experiment (Table 5).
It was noticed that untreated positive control rats suffered severe diarrhoea within the twenty four hours period following acetic acid per rectal infusion for induction of UC. This finding varied in intensity from mild to absent in all other groups, which denotes that acetic acid led to severe irritation.
Evaluation of the effects of pretreatments was performed by macroscopic examination of dissected colons by naked eyes (table 8), and by histopathologic examination (table 7) followed by immune-histochemical examination (table 9), and finally biochemical assay for detection of inflammatory markers (table 10), in addition to the qualitative test antineutrophil cytoplasmic antibodies(ANCA) which is specific for UC detection.
Macroscopic examination of colons dissected from negative control group showed intact mucosa with no signs of inflammation or haemorrhagic spots (score 0). Microscopic examination of mucosa of colons of rats in this group was normal and the lamina propria was normal with few eosinophils and normal crypts that were lined by mucin-secreting cells (fig 3a&b) , and both submucosa and T-muscularis(fig 4a&b)were also normal which was consistent with gross examination of negative control colons.
On the other hand colons dissected from untreated rats that received only acetic acid per rectum were severely ulcerated to the degree of perforation with grossly detected haemorrhagic areas in 100% of rats, which was also confirmed by histopathologic examination as severe deleterious histopathological lesions were demonstrated in the colon of Positive Control (C+ve) group, with increased pathologic lesion scoring. These histopathological lesions were characterized by diffuse ulcerative colitis with diffuse necrosis and desquamation of mucosal epithelium and complete necrosis as well as fragmentation of the crypts which are intensely infiltrated by neutrophils in addition to severe congestion of mucosal blood vessels (fig 3c) in addition to aggregation of bacterial colonies (fig 3d). The submucosa and tunica muscularis are greatly expanded by edematous fluids and neutrophilic cell infiltration (fig 4c & 4d, respectively). Liver showed mild granular degeneration of hepatocytes (fig 5b). Vacuolation of individual cells lining the renal tubules were demonstrated in the kidneys of this group (fig 6b).
On the other hand macroscopic examination of group treated with prednisolone which was used as a standandard drug revealed significant reduction in ulcer index as 62.5% of rats were affected and showed significant increase in percentage of ability of protection against UC (41.81%) compared to untreated group,the results were consistent with histopathology , which revealed pronounced improvement with significant decrease of pathologic lesion scoring, which revealed small multifocal ulcerative lesions with focal necrosis and desquamation of mucosal epithelium, focal mononuclear inflammatory cell infiltration (fig 3e) and few proprial hemorrhage (fig 3f). The submucosa and T.muscularis are infiltrated by few neutrophils (fig 4e & 4f, respectively). Mild focal vacuolar degeneration of hepatocytes was demonstrated in the liver (fig 5c), but normal renal tubules were demonstrated in the kidneys (fig 6c). .
In contrast to Prednisolone, gross examination by naked eye of group treated with leaves 250mg/kg revealed increased number and severity of ulcers in 87.5% of pretreated rats, with no significant improvement where the ability of protection against ulceration was only 15.1%, and that was confirmed by histopathology as there was diffuse necrosis of colonic mucosa associated with severe congestion of mucosal blood vessels and massive neutrophilic cell infiltration were frequently observed (fig 3g & 3h).In addition, intense infiltration of the submucosa with neutrophils was marked (fig 4g). The T.muscularis revealed marked separation of muscle fibers by edematous fluid and leukocytic cell infiltration (fig 4h). Swelling and vacuolation of hepatocellular cytoplasm were demonstrated in the liver (fig 5d). In addition, vacuolization of some renal tubular epithelial cells were demonstrated in the kidneys (fig 6d).
In comparison to low dose leave group, significant amelioration was recorded in the high dose leave group (500 mg/kg), by gross examination as the ulcer index was significantly reduced and the percent of protection of the high dose extract was 28.31% which was significantly higher than both low leave extract and positive control group as ulceration was detected only in 75% of pretreated rats, yet it was significantly less than prednisolone group. Consistently histopathologic examination revealed large focal erosive lesion and few proprial hemorrhage (fig 3i & 3j). But the sub mucosa and T.muscularis were intensely infiltrated with neutrophils (fig 4i & 4j, respectively). The liver and kidneys of this group appeared normal (fig 5e & 6e, respectively).
The group pretreated with pseudostem 250 mg/kg showed better macroscopic examination profile as the number and severity of ulcers was less as they were detected in only 75% of pretreated rats which consequently significantly reduced the ulcer index compared to leaves pretreated group by low and dose and also to positive control group, but its protective effect was significantly less than prednisolone and almost the same as leaves pretreated group with high dose as the % of protection was 28.52 % in pseudostem low dose group .The histopathologic examination of pseudostem low dose revealed pronounced attenuation of the pathological lesions with decreases pathologic lesion scoring,small focal necrosis of mucosal epithelium associated with mild proprial edema and few leukocytic cell infiltration were demonstrated (fig 3k & 3l). Mild infiltration of submucosa and T.muscularis with neutrophils was recorded in this group (fig 4k & 4l, respectively). Normal histological structures of liver and kidneys were also demonstrated (fig 5f & 6f, respectively).
Regarding the gross examination of the high dose of pseudostem (500 mg/kg) and low dose of fruit extract (250 mg/kg), ulcers were detected in only 62.5% ,which is the same percentage of affected rats in the prednisolone (standard),also the number and severity of ulcers were approximately close to each other leading to non significant differences in ulcer indices and consequently % of protection of both pseudostem extract high dose (40.34%) and fruit extract low dose (41.13%)on one side, and prednisolone (41.81%) which is the standard treatment on the other side, however their protective effects were significantly higher than those of leaves low and high doses as well as pseudostem low dose, and of course the positive control group. On the other hand they showed significant lower protective effects than fruits extract high dose (500 mg/kg), whose protective effect was the highest of all pretreatments when compared to positive group and each pretreatment with pronounced significant % of protection of 53.33% and least number of ulcers, severity and affection of only 50% of rats, consequently exhibiting the least ulcer index among all other groups. The histopathologic photomicrographic findings were consistent with the macroscopic examination of these groups, where normal colonic mucosa in most examined sections was frequently demonstrated in high dose stem-treated groups stem group. Regenerative activity of the mucosal epithelium and minimal leukocytic cell infiltration as well as scant proprial hemorrhage were demonstrated in high dose stem-treated groups (fig 3m & 3n).The submucosa was infiltrated with few neutrophils (fig 4m) and the T.muscuaris showed edema with few neutrophilic cell infiltration (fig 4n). Normal hepatocytes and renal parenchymal structures were also demonstrated (fig 5g & 6g, respectively). Much better improvement, with marked regenerative activity of the colon mucosa and proliferation of colonic lymphoid nodules and minimal leukocytic cell infiltration were recorded in the mucosa of low dose fruit treated groups (fig 3o & 3p) and high dose fruit treated groups (fig 3q & 3r). The submucosa and T.muscularis of low dose fruit treated groups were mildly infiltrated with neutrophils (fig 4o & 4p). Only mild focal congestion of some hepatic sinusoids were demonstrated in this group (fig 5h), but normal histological structures were demonstrated in the kidneys (fig 6h) Sparse neutrophils were demonstrated in the submucosa and T.muscularis of high dose fruit treated groups (fig 4q & 4r). Normal heptic and renal parenchyma were demonstrated (fig 5i & 6i, respectively).
The results of MPO immune-histochemical expression recorded in the colonic mucosa and submucosa showed that individual MPO+ cells were demonstrated in the mucosa and submucosa of the colon of normal rats (fig 7a & 8a). Whereas, increased expression of MPO with significant increase of % of MPO+ cells was recorded in the colonic mucosa and submucosa of C+ve group (fig 7b & 8b). Significant decrease of % of MPO+ cells was recorded in the mucosa and submucosa of Prednisolone-treated group (fig 7c & 8c). The colon of low dose leave group showed increased % of MPO+ cells, which are insignificantly different from the C+ve group, in the mucosa and submucosa (fig 7d & 8d). But significant difference was recorded in the high dose leave group in both the mucosa and submucosa (fig 7e & 8e). Better improvement with marked decrease of % of MPO+ cells was recorded in the mucosa (fig 7f & 7g) and submucosa (fig 8f & 8g) of low and high dose stem-treated groups, with insignificant difference between them. On the other hand, significant difference was recorded between low and high dose fruit treated groups. Remarkable decrease of MPO expression with significant decrease of MPO+ was recorded in the mucosa and submucosa of low dose fruit treated groups (fig 7h). Only few scattered MPO+ cells were demonstrated in the mucosa and submucosa of high dose fruit treated groups (fig 7i & 8i), while low dose fruit treated group showed significant decrease of MPO+ cells with brown staining (fig 8h).
Biochemical analysis of sera obtained from rats infused per rectally with acetic acid in this study aiming at inducing a rat model of UC ,revealed highly significant elevation of inflammatory markers CRP and Ilβ6 in the untreated group positive control compared negative control and to all other treated groups. The degree of inflammation was variable between the treated groups, all showed significant protection but the highest were those of the groups given prednisolone and fruit extract in high dose as these groups showed non significant difference from the negative control group.
Regarding ANCA test which is a highly specific qualitative test for diagnosis of UC, it revealed 100% negativity in the negative control group and in all treated groups except leaves low dose where it was 75% negative. On the contrary, the positive control results were 100% positive. This finding is in enforced by Pang et al,  in their study as they stated that ANCA test is diagnostic for UC and its quantification reveals the severity of UC.
The anti-inflammatory effects of phenolics present in Musa extract when they were given orally in our study are due to serial enzymatic reactions that take place in the digestive system. After being absorbed in the small intestine they conjugate with glucuronic acid and sulfonate, then 5%-10% pass to the plasma , but the largest portion (90–95%) pass directly to the large intestine (colon) . where fermentation occurs by colonic microbiota ,leading to elaboration of the positive effect of phenolics on colon’s health by reducing its pH as anti-inflammatory effects ,which was emphasized in our study and consequently suppression of cancer cells.
In the present study an animal model mimicking Ulcerative colitis was induced by using per rectal acetic acid infusion, it produced severe inflammation which could be prohibited by pretreatment with natural plant extracts rich in Flavonoids and phenolic acids due to their protective effects on the colon as 90-95% of phenolic acids are metabolized in the colon by microorganisms that lead to their anti-inflammatory activity.
This was clear in our study as characterization of secondary metabolites in Musa revealed high contents of these compounds.
It is noteworthy mentioning that the Musa fruits had the best effect regarding the ability to protect against development of severe Ulcerative colitis ,which introduces a promising natural supplement that can be used in future clinical studies for further evaluation of its effect as a protective pretreatment in vulnerable patients that are susceptible to have Ulcerative colitis.