Anti-ulcerative colitis effect of Calligonum comosum L'Hér. using rat model: Chemical, pharmacological and histopathological evidences

Abstract

Calligonum comosum is a perennial shrub growing and widely used in traditional medicinal system in Saudi Arabia. The total phenolic content and in vitro antioxidant activity were compared between the water extract (WE) and methanol extract (ME). The protective potential against acetic acid (AA) induced ulcerative colitis (UC) was also evaluated in rats. The obtained results showed that the total phenolic content of the WE and ME were 8.378 ± 0.738 and 33.819 ± 0.488 µg/mL. The antioxidant properties of the two extracts were directly influenced by their total phenolic contents. The ME with higher phenolic contents and stronger antioxidant power was more effective than the WE in protection against AA-induced colitis. Phytochemical study of the ME led to the identification of three flavonoid derivatives: (-)-epi-catechin, quercetin-3-O-α-l-arabinofuranoside (Avicularin) and quercetin-3-O-β-d-glucuronide-6″-methyl ester by various spectroscopic methods. (-)-Epi-catechin was the major component while the other two compounds were obtained in minute quantities. The anti-ulcerative colitis effect of the ME can be explained by the presence of the antioxidant flavonoids since AA-induced colitis featured by imbalance between oxidant and antioxidant substances. Further support of such explanation was provided by HPLC quantification of (-)-epi-catechin in the ME and WE. The percentage in ME was higher than the WE but the difference was higher in term of Total Phenolic Content (TPC). These results support the traditional use of C. comosum as anti-ulcerative colitis.

1. Introduction

Calligonum L. genus characterized by xeromorphic shrubby members and comprises more than 80 species allocated throughout Southern Europe, Northern Africa and Western Asia (Brandbyge, 1993). This genus of the family Polygonaceae are mainly 3 m height evergreen shrub. Calligonum comosum members are woody evergreen plants present in the Arabian Sahara characterized by sandy soils (Ozenda, 1991). The fresh flowers are consumed as food as they are rich in nitrogenous and sugar components (Taia and Moussa, 2011). The plant is used in folk medicine to manage several ailments including hepatotoxicities, polycystic ovary and osteoporosis (Barati and Movahedin, 2021). C. comosum extract is used as anti-inflammatory and anti-ulcer agent. Further, the plant is used by some local healers to overcome stomach ailments; curing toothache, as stimulant and astringent (Liu et al., 2001). The plant is reported to possess cytoprotective and hypoglycemic activities (Liu et al., 2001, El-Hawary and Kholief, 1990).

The ethyl acetate fraction obtained from the methanol extract was the most active as anticancer (Badria et al., 2007). Various C. comosum extracts were effective anti-Listerial species causing food contamination (Riadh et al., 2011). C. comosum produces secondary metabolites including phenolics, tannin, flavonoids, alkaloids, steroids, terpenoids and saponins (Cheruth et al., 2016). Phytochemical study of the plant chloroform extract let to the isolation of coumaric acid, 4-ethoxy-1,2-benzendiol and tamgermanetin, while the ethyl acetate fraction of C. azel afforded 5-(p-hydroxy-phenyl)-pent-2-en-oic acid, phlorizin, protocatechuic acid, methyl gallate, caffeic acid, 5-(p-hydroxy-phenyl)-3-hydroxy-pentanoic acid and α,β-diamino-4-hydroxybenzenebutanoic acid (Belaabed et al., 2017). GC–MS/MS-based phytochemical analysis of C. polygonoides flower bud methanol extract led to the identification of 93 compounds including phenols, monoterpenes fatty alcohols and fatty acids (Berwal et al., 2021).

The current study was conducted to provide scientific evidences for the use of C. comosum as anti-ulcer agent. Chemical, pharmacological, histopathological and analytical evidences were provided.

2. Material and methods

2.1. Plant material

Collection of Calligonum comosum L'Hér. plants from Al Sir Desert across, Al Qassim region was done in the spring season 2020. The plant material was identified by Prof. Saniya Kamal, Botany Department, College of Science, Alexandria University. The specimen # 19,420 was preserved in the Department of Botany herbarium at the College of Science, Alexandria University. The plant materials were dried in shade in well aerated room under controlled temperature 21 ± 1 °C.

2.2. Extraction

The dried powdered plant materials (1 kg) were extracted using methanol (99.8 %) (4 × 5L) at room temperature. The combined extracts were dried under Vacuum to produce 103 g of the methanol extract (ME). Extraction by decoction was applied to another 200 g of the plant material with reflux for 4 h. The resulted decoction was dried using Freeze dryer instrument to produce 24 g lyophilized water extract (WE).

2.3. Total phenolic content (TPC)

Total Phenolic Content (TPC) was determined in ME and WE using Folin–Ciocalteu test (Johari and Khong, 2019). The extracts were prepared as solution in water/methanol with 1 mg/mL concentration. About 100 μL of the solutions were mixed with 0.75 mL of the Folin–Ciocalteu reagent (diluted 10-fold with deionized water). The mixtures were allowed to settle for 5 min at a room temperature followed by adding 0.75 mL of sodium carbonate (Na₂CO₃ 7.5 %) mixing well by shaking. The absorbance of the final sloutions was measured at 725 nm after 90 min.

2.4. In vitro antioxidant assays

2.4.1. DPPH radical scavenging assay

The DPPH radical scavenging power of C. comosum ME and WE were measured using the reported method (Kedare and Singh, 2011). Percentage of the radical scavenging activity of DPPH was obtained applying the following formula:

$$\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}-\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}\mathrm{x}100$$

2.4.2. Ferric-reducing power assay

The ferric chloride reducing ability of the C. comosum extracts and fractions were determined as previously reported (Benzie and Strain, 1999).

2.5. In vivo experiments

2.5.1. Experimental animals

Male Wistar rats of about 180–200 g were used. Animals were housed in cages with ventilation system (Rat IVC Blue Line, Techniplast, Buguggiate VA, Italy) at the lab animal unit, College of Pharmacy; Prince Sattam Bin Abdulaziz University (PSAU). The animals were kept under controlled conditions of 12 h/12 h light/dark cycle, 25 ± 1 °C and with feed and water ad libitum. All the preformed procedures complied with the internationally adopted guidelines (NRC, 2011). The procedures were performed under the Bioethical Research Committee (BERC) approval at PSAU (Ref No. BERC-008–04-21).

2.5.2. Protective effect against AA–induced colitis in rats

The anti-ulcerative effects of ME and WE were evaluated in rats using AA–induced colitis model (Mascolo et al., 1995). Animals were randomly divided into following groups of 5 rats in each:

Group I: Normal control rats (NC) received vehicle (5 mL/kg).

Group II: Colitis control rats (CC) received vehicle (5 mL/kg).

Group III: Colitis rats received the reference drug; prednisolone sodium phosphate (REF) at 5 mg/kg.

Groups IV and V: Colitis rats received ME at 250 mg/kg (ME-250) and 500 mg/kg (ME-500), respectively.

Groups VI and VII: Colitis rats received WE at 250 mg/kg (WE-250) and 500 mg/kg (WE-500), respectively.

Reference drug and extracts were prepared using 0.2 % Tween 80 in normal saline shortly before the administration. All treatments were administered orally, once daily for 7 days. The last dose was administered and 2 h later colitis was induced.

2.5.3. Induction of experimental colitis

The animals were fasted for 16 h with free access to water before the induction of colitis. Diethyl ether was used for rat’s anesthetization, and a flexible polyethylene catheter was inserted into the anus for about 8 cm. Two mL of AA (4 % v/v in 0.9 % saline) were slowly administered. The rats were hold in a trendelenburg position to avoid solution leakage for half minute. The colons were then washed with excess of saline (2 mL). The rats were scarified 48 h later by cervical dislocation, and laparotomy was performed. The last 8 cm of the colons were excised, longitudinally opened and rinsed with normal saline. The colon specimens were weighed and the ratio of wet weights to the lengths (w/L) were calculated (Appleyard and Wallace, 1995).

The degree of colon damage was graded on a 0–5 scale as reported earlier (Buell and Berin, 1994). The scoring system was done as: Score 0 = no damage; Score 1 = localized hyperemia; Score 2 = erosions or ulcers with absence of inflammation; Score 3 = erosions or ulcers with single inflammation; Score 4 = two or more sites of ulceration and/or inflammation and Score 5 = two or more major inflammation and ulceration sites or one major inflammation and ulceration site more than 1 cm along the colons length. Furthermore, ulcer areas were measured by tracing margins of the ulcers with the help of transparent sheet using graph sheet. Each cell on the graph sheet was 1 mm² in area and the cells number was counted to get the ulcer area for each colon. For each specimen, ulcer index was calculated using the following formula (Minaiyan et al., 2008):

$$\mathit{Ulcer}index=Ulcerarea\left(c{m}^2\right)+Macroscopicdamagescore.$$

2.6. Histopathological evaluation of colon tissues

Neutral buffered formalin 10 % was used to fix small portions of colons specimens for 24 h. The tissues were dehydrated, embedded in paraffin, and 5 µm thickness sections were obtained. The obtained sections were then stained using Hematoxylin and Eosin (H&E), Periodic acid-Schiff reactive (PAS) and Alcian blue for 5 min. The slides were extensively examined under light microscope (Olympus, Japan) to detect any pathological changes. (Hamad and Ahmed, 2018).

2.7. Chromatographic purification

Part of the ME (20 g) was subjected to fractionation over Sephadex LH20 column eluted with CHCl₃/Acetone 3:1 (500 mL), CHCl₃/Acetone 1:3 (1L), Acetone/MeOH 9:1 (1L), Acetone/MeOH 3:1 (500 mL), Acetone/MeOH 1:1 (500 mL)and 100 % MeOH (500 mL). Fractions of 100 mL were screened with TLC then, fractions with same profile were pooled together to yield five fractions I-V. Part of fraction III (8 g) was further purified using column chromatography (500 g silica gel, Loba Chemie PVT. ltd. 100–200 Mesh). Elution started with EtOAc then EtOAc/MeOH mixtures with gradual raising in the percentage of MeOH in a gradient elution system. Fractions of 250 mL were collected, checked by TLC then, chromatographically homogenous fractions were combined together to end up with ten fractions A- I.

Fraction A (3.2 g) eluted with ethyl acetate was further fractionated over 200 g silica gel column (Merck, Mesh size 70–230, 3 cm i.d.) starting with EtOAc then EtOAc/MeOH mixtures with increasing the ratio of MeOH in a gradient elution. Fractions of 50 mL each were gathered, explored by TLC and fractions of similar profile were pooled. Fraction A- 4 (1.88 g) was subjected to MPLC RP18 (40–63 µg particle size) column (920 mm, 46 mm i.d., Buchi, Borosilikat 3.3 Code No. 44835) eluting with water followed by water/MeOH mixtures in a gradient system (1.8 L). Fractions of 15 mL each were obtained and fractions of similar pattern on TLC were collected. Fractions eluted with 60 % MeOH afforded 125 mg of 1.

Fraction B (879 mg) was subjected to MPLC RP18 (40–63 µg particle size) column (920 mm, 46 mm i.d., Buchi, Borosilikat 3.3 Code No. 44835) eluting with water followed by water/MeOH mixtures in a gradient system (1.8 L). Fractions of 15 mL each were gathered, screened by TLC and similar fractions were collected. Fractions eluted with 40 % MeOH (50 mg) were further purified with RP18 PTLC developed with 40 % water in MeOH to afford 7 mg of 2 and 4 mg of 3.

2.7.1. Compound 1

C₁₅H₁₄O₆. Solid; [α]²⁰_D −82° (c 1.0, MeOH). UV λ^max (MeOH) 263 nm; ¹H and ¹³C NMR: Tables S2, S3. HRESIMS: 289.0723 (Cal. 289.0712) [M⁺−H], 291.0882 (Cal. 291.0869) [M⁺+H].

2.7.2. Compound 2

C₂₀H₁₈O₁₁.Yellow solid; UV λ^max (MeOH) 354, 257; (NaOMe) 396, 323, 273; (AlCl₃) 435, 354, 275, 250; (AlCl₃/HCl) 402, 364, 271, 246; (NaOAc) 378, 323, 273, 248 nm; ¹H and ¹³C NMR: Tables S2, S3. HRESIMS: m/e 867.1634 (Cal. 867.1620) [2 M⁺−1], 433.0779 (Cal. 433.0771) [M⁺−1], 435.0920 (Cal. 435.0927) [M⁺+1], 457.0738 (Cal. 457.0747) [M⁺+Na], 303.0497 (Cal. 303.0505) [M⁺−sugar].

2.7.3. Compound 3

C₂₂H₂₀O₁₃.Yellow solid; UV λ^max (MeOH) 353, 258; (NaOMe) 390, 320, 274; (AlCl₃) 433, 359, 275, 251; (AlCl₃/HCl) 403, 323, 273, 248; (NaOAc) 374, 320, 273 nm; ¹H and ¹³C NMR: Tables S2, S3. HRESIMS: m/e 983.1742 (Cal. 983.1739) [2 M⁺−1], 491.0835 (Cal. 491.0826) [M⁺−1], 493.0981 (Cal. 493.0982) [M⁺+1], 515.0801 (Cal. 515.0802) [M⁺+Na].

2.8. HPLC quantification of 1 in ME and WE

2.8.1. Equipment and instruments

The Liquid chromatographic analysis of pure (-)-Epi-catechin, ME and WE were performed using UHPLC (Dionex UltiMate 3000, Thermo Fisher Scientific®), fitted with diode array detector (DAD–3000; Thermo Fisher Scientific®), quaternary pump, degasser and autosampler (Dionex UltiMate 3000, Thermo Fisher Scientific®). Data were analyzed and processed under the control of the software Chromeleon 7.2.8 developed by Dionex, Thermo Fisher Scientific®. The quantifications were performed at wavelengths of 280 nm. Separation was obtained using a C₁₈ column (250 mm × 4.6 mm i.d., particle size 5 µm, WATERS®) at room temperature with5 µL injection volume of the different concentrations of (-)-epi-catechin, ME and WE. Ultrapure water (A) and acetonitrile (B) both acidified with 0.1 % acetic acid, under flow rate of 1.0 mL/min were used as mobile phase. A gradient system starting with zero percent of B followed linear gradient run from 100 % (A) to 15 % (B) within 5 mins. The percentage of (B) increased to 25 %, 50 % and 100 % after 20, 25 and 27 min, respectively. re-equilibration for 3 min to initial composition to complete the run of 30 min.

2.8.2. Method validation parameters

The developed method was validated as advised by the International Conference on Harmonization (ICH) (ICH, 2005).

2.8.2.1. Calibration curve

Calibration curve was obtained from the chromatographic analysis of (-)-epi-catechin (1) methanol solutions isolated in the study. Solutions containing (5; 20; 100; 250, 500 and 1000 µg/ml) were prepared from a stock solution of 10 mg/10 mL by dilution using HPLC grade methanol. Injections were done in triplicate. Regression equations were obtained as correlation between (Y) the peak areas and (X) the sample concentrations.

2.8.2.2. Linearity

(-)-Epi-catechin (1) concentrations starting from 5 to 1000 μg/ mL were used to obtain the calibration graph between peak area versus concentration. The linearity and correlation coefficient (R²) were obtained using the least square regression equation.

2.8.2.3. Accuracy

(-)-Epi-catechin (1) (0, 50, 100, and 150 %) were added to samples with previously quantified amounts and the mixtures were reanalyzed to determine the recovery of the proposed method.

2.8.2.4. Precision

To evaluate precision, repeatability as well as intermediate precision were determined. Repeatability of the HPLC method was proved by performing six independent analyses of (-)-epi-catechin followed by calculation the relative standard deviation percentage (RSD). Intra‑ and inter‑day precisions were accessed by repetition of the analysis on one day (intra‑day precision) then on the three following days (inter‑day precision). For each analysis the %RSD was determined.

2.8.2.5. Robustness

The method’s robustness was proved by intentionally introduce slight alteration in the chromatographic conditions and observing the impact on the analysis results. Variations in the detection wavelength was enforced and impact on peak area was observed.

2.8.2.6. Limit of detection and limit of quantification

Limit of detection (signal‑to‑noise ratio = 3.3) (LOD) as well as limit of quantification (signal to noise ratio = 10) (LOQ) has been determined as per the ICH guidelines.

2.9. Statistical analysis

The data were expressed as mean ± SEM. The results were analyzed by SPSS version 19 using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. Graphical representations are created by Microsoft Excel 2010. The variations between the mean values were statistically significant when P < 0.05.

3. Results and discussion

Two extracts obtained by different extraction methods and solvents (ME and WE) of the plant materials were compared for their TPC, antioxidant and anti-UC effects. The ME extract showed better effect in all the tested parameters. Trying to correlate the activity with the chemical components the ME was subjected to several chromatographic purifications directed by TPC to identify the major active and two minor compounds.

Reports on the chemical components of Calligonum species indicated that they are rich in phenolic derivatives (Riadh et al., 2011, Cheruth et al., 2016). The various traditional uses and biological activities are most likely due to the phenolic contents. Berwal et al. correlated the value of C. polygonoides in the management of some chronic diseases mainly to the phenolic contents such as 2,3-dihydro-2,5dihydroxy-6-methyl-4H-pyran-4-one (DDMP), furan-2,5-dimethyl, deoxyspergualin, 2-methoxy-4 vinylphenol, dehydromevalonic lactone, benzeneethanol-4-hydroxy and quinic acid (Berwal et al., 2021). The TPC were determined according to the reported method (Johari and Khong, 2019) in the ME and WE and Sephadex LH20 column fractions. A calibration curve of reference standard gallic acid ranging from 6.25 to 50 µg/mL was obtained and the TPC were presented as milligrams per 100 g of gallic acid equivalents in the ME and WE. The ME was found to have about 3 folds more TPC than the WE (Table S1).

The in vitro Antioxidant activity applying the DPPH radical scavenging (Kedare and Singh, 2011) and Ferric-reducing power assays (Benzie and Strain, 1999) were performed on ME and WE. The antioxidant activity of C. comosum ME and WE were evaluated for the ability to suppress the DPPH, which is one among few stable organic nitrogen radicals. When reduced by an antioxidant compounds DPPH undergoes colour changed from purple to yellow. All tested extracts and fractions resulted in dose dependent response’s. The relative potencies of the different tested materials are presented in Fig. 1A. None of the tested fractions was more active than ascorbic acid. The results showed that the WE was very ineffective.

Fig. 1.

Comparative DPPH radical scavenging activity (A) and Ferric-reducing power (B) of the ME and WE of C. comosum.

The reducing power capacity of C. comosum ME and WE were explored as their ability to cause the reduction of ferric (Fe³⁺) to ferrous (Fe²⁺) form Perl’s Prussian blue. A concentration dependent response was obtained in all tested samples. The relative potencies of the different tested materials are presented in Fig. 1B.

UC is a common inflammatory bowel disease resulted in destruction of the integrity of the colon accompanied by clinical signs as diarrhea and rectal bleeding. It also leads to macroscopic alterations in the colon such as ulceration and edema (Matsuoka et al., 2018). Acetic acid (AA)-induced colitis is a well-established, easy and widely used model for studying UC (Randhawa et al., 2014). This protocol results in enhanced vasopermeability and infiltration of neutrophils and macrophages to the affected colon indicating the induction of inflammatory condition (Popov et al., 2006). Previous studies stated that the presentation of AA-induced colitis in experimental animal’s mimics acute human intestinal inflammation (Randhawa et al., 2014, Low et al., 2013).

The W/L ratio is used as an indicator of inflammation. The severity of UC, the severity of inflammation, swelling and thickening in the intestinal walls following ulceration can be reflected by the significant elevation in the colon W/L ratio (Thippeswamy et al., 2011, Rachmilewitz et al., 1989). W/L ratio also suggest the inflammatory nature of AA-induced UC. The colon tissues of rats exposed to AA exhibited a marked increase in colon W/L ratio compared with the NC group (Table 1, p < 0.05). Conversely, the colon of rats pre-treated with the reference drug (prednisolone sodium phosphate), ME-250 and ME-500 showed significant (p < 0.05) improvements in colon W/L ratio when compared to the CC. Both doses of WE had no marked effect on colon W/L ratio compared to CC rats.

Table 1.

Prophylactic effect of the water extract (WE) and methanol extract (ME) of Calligonum comosum on the macroscopic parameters of colitis induced by acetic acid in rats.

Groups	W/L ratio (g/cm)	Lesion score (0–5)	Ulcer area (cm2)
NC	0.25 ± 0.01	0.00 ± 0.00	0.00 ± 0.00
CC	0.58 ± 0.04●	4.8 ± 0.20●	6.72 ± 0.57●
REF	0.32 ± 0.01●#	1.8 ± 0.20●#	1.54 ± 0.15●#
ME-250	0.39 ± 0.02●#ϕ	2.6 ± 0.24●#ϕ	2.08 ± 0.07●#ϕ
ME-500	0.36 ± 0.02●#	2.2 ± 0.20●#	1.86 ± 0.11●#
WE-250	0.53 ± 0.02●ϕ	4.2 ± 0.37●ϕ	6.28 ± 0.52●ϕ
WE-500	0.49 ± 0.01●ϕ	4.0 ± 0.32●ϕ	5.50 ± 0.31●ϕ

Values are tabulated as the mean ± S.E.M., n = 5 rats/group.

● indicates significant difference versus normal control (NC) group at p < 0.05.

# indicates significant difference versus colitis control (CC) group at p < 0.05.

ϕ indicates significant difference versus reference (REF) group at p < 0.05.

Visible signs of colon tissue inflammation such as localized hyperemia, erosions, inflammation and ulceration were quantified as a lesion score ranging between 0 and 5 in ascending order of severity. The colon tissues of the CC group displayed a significant elevation in the value of ulcer score along with an increase in the values of ulcer area and ulcer index (Fig. 3) when compared against the NC group. The increases in these values with marked erythema of the colon wall demonstrated the mucosal damage and ulceration due to AA (Dey et al., 2017). Macroscopic assessment of colon samples (Fig. 2, Fig. 3) revealed that the CC group had severe signs of inflammation and bleeding accompanied by significantly higher values of lesion score, ulcer area and ulcer index than the NC group. These values were remarkably decreased in animals treated with REF prednisolone sodium phosphate, ME-250 and ME-500 compared with the CC group. Administration of WE-250 and WE-500 did not induce and significant improvements in the observed macroscopic parameters compared with the CC group (Table 1 and Fig. 2, Fig. 3). Consequently, pretreatment with ME significantly reduced the colon mucosal damage, inflammation and histological alteration induced by AA confirmed by comparison with the standard drug, prednisolone sodium phosphate. Interestingly, ME-500 was able to improve the colon W/L ratio similar to those of the reference group. Previous studies reported the identification of several phytochemicals in the methanol extract of C. comosum such as phenolic compounds (Cheruth et al., 2016). Accordingly, the protective effect of ME may be due its phenolic contents.

Fig. 3.

Effects of C. comosum extracts on ulcer index induced by acetic acid in rats. Values are expressed as mean ± S.E.M., n = 5 rats/group. ● indicates significant difference versus NC group at p < 0.05. # indicates significant difference versus CC group at p < 0.05. ϕ indicates significant difference versus REF group at p < 0.05.

Fig. 2.

Representative photographs of colon tissue 2 days after AA–induced colitis in rats: (A) NC group treated with the vehicle; (B) CC group treated with the vehicle; (C) colitis group treated with prednisolone sodium phosphate; (D & E) colitis groups treated with ME-250 and ME-500, respectively; (F & G) colitis groups treated with WE-250 and WE-500, respectively.

Literature survey indicated that three histological methods namely Hematoxylin and Eosin (H&E), Periodic Acid-Schiff (PAS) and Alcian blue (AB) were used to assess UC. H&E stain provides a comprehensive picture of the tissues. PAS is commonly used to detect neutral tissue mucins, and detects other tissue components including glycoproteins and glycolipids (Meyerholz et al., 2018). Further, AB is used to stain acidic mucins (Dong et al., 2012). In the current study H&E (Fig. 4), Periodic acid-Schiff (Fig. 5) and Alcian blue (Fig. 6) stains were used to evaluate the architectures of the colon tissues. Colon tissue of the NC group displayed as expected normal histological architecture including normal mucosa with intact epithelium, simple tubular colonic crypts lined and extended down to the muscularis mucosae (Fig. 4-A). The goblet cells producing neutral mucin showed normal appearance (Fig. 5-A). Further, goblet cells that are producing acid mucin showed normal distribution (Fig. 6-A). Conversely, histopathological analysis of H&E stained colons in the CC group demonstrated marked destruction of crypt structure with multiple areas of necrosis, a disturbed epithelial layer and thickening of basement membrane (Fig. 4-B). PAS-stained specimens of CC rats showed sever reduction in functioning goblet cells that are producing neutral mucin (Fig. 5-B) while Alcian blue stain showed absence of acid mucin (Fig. 6-B).

Fig. 4.

Representative photographs of colon tissue 2 days after AA–induced colitis in rats: [A]: NC group treated with the vehicle; [B]: CC group treated with the vehicle; [C]: colitis group treated with prednisolone sodium phosphate; [D & E]: colitis groups treated with ME-250 and ME-500, respectively; [F & G]: colitis groups treated with WE-250 and WE-500, respectively.

Fig. 5.

Histological sections of rat colon after treatment with ME and WE extracts at 250 and 500 mg/kg using Periodic acid Schiff (PAS) stain, magnification x400, and scale bar 20 µ. [A]: shows normal PAS appearance of goblet cells (G) producing neutral mucin (magenta color) in abundant amounts, [B]: shows very few numbers of functioning goblet cells (G) that are producing neutral mucin (magenta color) and suffering from absence of functioning goblet cells (A), [C]: Almost normal in amount and distribution of goblet cells that are producing neutral mucin (G), [D]: shows very slightly improvement because very few goblet cells (G) restore their function of producing neutral mucin while most of mucosa suffering from absence of functioning goblet cells (A), [E]: shows much improvement by restoring function of producing neutral mucin from goblet cells (G), and almost like normal, [F]: shows very slightly improvement with very few areas of restoring function of producing neutral mucin by goblet cells (G), [G]: shows slightly improvement with few areas of restoring function of producing neutral mucin by goblet cells (G).

Fig. 6.

Histological sections of rat colon after treatment with ME and WE extracts at 250 and 500 mg/kg using Alcian blue stain, magnification x400, and scale bar 20 µ. [A]: shows normal distribution of goblet cells that are producing acid mucin (blue color) in abundant amounts, [B]: shows intestinal tissue with large areas suffering from almost completely absence (A) of functioning goblet cells which is indicated by absence of acid mucin, [C]: Almost normal in amount and distribution of goblet cells (G) that are producing acid mucin, [D]: shows moderate improvement by moderate restoring functioning goblet cells (G) that are producing acid mucin (blue color), [E]: shows excellent improvement by almost restoring normal functioning goblet cells (G) that are producing acid mucin (blue color), [F]: shows very slightly improvement and very few numbers of goblet cells (G) restored ability of producing acid mucin (blue color), [G]: shows moderate improvement by moderate restoring functioning goblet cells (G) that are producing acid mucin (blue color).

Colon tissue from REF treated rats exhibited predominantly normal histology of the colon tissues with maintained epithelial layer and crypt structure when compared to CC group (Fig. 4-C). The goblet cells producing neutral mucin (Fig. 5-C) and those producing acid mucin (Fig. 6-C) showed almost normal distribution. ME pretreatment exerted a dose–dependent protective effect against the histopathological changes in the rat colons induced by AA. Rats pretreated with 250 mg/kg/day ME expressed moderate inflammatory cell infiltration with moderate focal necrosis was observed (Fig. 4-D). The colon tissues showed improvement in the function of neutral mucin producing goblet cells (Fig. 5-D) in addition to moderate restoring functioning goblet cells that are producing acid mucin (Fig. 6-D). Best results were obtained in specimen of rats treated with ME at 500 mg/kg as they showed a close similarity to the REF group. Almost normal sections were observed in H&E-stained specimens (Fig. 4-E). In PAS, much improvement by restoring function of producing neutral mucin from goblet cells (Fig. 5-E). Further, acid mucin secretion was restored to almost normal as indicated by Alcian blue (Fig. 6-E). Conversely, histopathological study of the WE-250 treated group suffered from severe inflammatory cell infiltration with marked necrosis, edema and hyperemia (Fig. 4-F), as well as severe goblet cell depletion which is indicated by absence of neutral mucin producing goblet cells (Fig. 5-F) and acid mucin producing goblet cells (Fig. 6-F). In general, similar histopathological changes were observed in the distal colon of WE-500 treated rats (Fig. 4-G, 5-G, 6-G).

The ME was superior compared with the WE in all the chemical and pharmacological parameters. To follow component’s responsible for the activity the ME was subjected to chromatographic purification on Sephadex LH20 to afford fractions I-V. Fraction III showed the highest TPC (79.154 ± 0.825 mg GAE/g) and was subjected to chromatographic purification on silica gel to obtain one major compound 1 and two minor compounds 2 and 3. The HRESIMS (Material and Methods, Fig. S11) of 1 showed ion at m/e 289.0723 [M⁺−H] and 291.0882 [M⁺+H] for the molecular formula C₁₅H₁₄O₆. ¹H NMR of 1 in acetone‑d₆ (Material and Methods, Fig. S1) was diagnostic for 3,4-dihydro-2-(3,4-dihydroxyphenyl)–2H-chromene-3,5,7-triol known as catechin or one of its isomers. Ring C spin system at δ_H 4.59 (d, J = 7.8), δ_C 82.54 (CH); δ_H 4.03 (d, J = 4.0), δ_C 68.32 (CH) and δ_H 2.56 (dd, J = 8.4,16.0), 2.94 (dd, J = 5.6,16.0), δ_C 28.68 ppm (CH₂) indicated one of the epi-catechin stereoisomers (Abd El-Razek, 2007). The negative sign in optical rotation enable the identification of 1 as (-) epi-catechin (Fig. 7).

Fig. 7.

Chemical structures of 1–3.

The UV spectra (Material and Methods) of 2 and 3 in MeOH indicated the presence of 5,7, 3′,4′-tetrahydroxyl substituted flavonols (Mabry et al., 1970). The ¹H NMR of the two compounds (Fig. S12-S14, S24-S27, Table S2) showed ABX systems for ring B protons and two meta coupled doublets for ring A protons. Both ¹H NMR and ¹³C NMR data (Fig. S15- S19) of 2 showed signals for a pentose moiety diagnostic for arabinofuranoside located at C-3. Final evidence for the structure of 2 was obtained from HRESIMS (Fig. S23) where it shows 2 M⁺−1 at m/e 867.1634, M⁺−1 at m/e 433.0779, M⁺+1 at m/e 435.0920, M⁺+Na at m/e 457.0738 and an ion peak at m/e 303.0497 representing the aglycone + H. The data of 2 is quite similar to those reported for quercetin-3-O-α-l-arabinofuranoside also known as “Avicularin” isolated from Psidium guajava (Metwally et al., 2010). ¹³C NMR, DEPT 135 and APT spectra of 3 (Fig. S28-S31, Table S3) indicated the presence of 5 oxygenated CH and one carbonyl at δ_C 170.83 ppm. These data of the sugar moiety were identical with those reported for glucuronylpyranoside located at C-3. The position of the glucuronylpyranoside at C-3 was supported by the HMBC (Fig.S24) 3-bonds correlation of H-1’’ of the sugar at δ_H 5.24 (d, J = 7.8 Hz) with the C-3 at δ_C 136.74 ppm. ¹H NMR and ¹³C NMR data (Fig. S24- S31) also showed methoxyl group signals at δ_H 3.67 (bs) and δ_C 53.01 ppm. The position of the methoxyl group assigned to C-6” of the glucuronylpyranoside based on the HMBC (Fig. S24) 3-bonds correlation between the methoxyl proton signal at δ_H 3.67 and the C-6” at δ_C 170.83 ppm. Supporting evidence for the structure of 3 was obtained from HRESIMS (Figure S41) where it shows 2 M⁺−1 at m/e 983.1742, [M⁺−1] at m/e 491.0835, [M⁺+1] at m/e 493.0981 and [M⁺+Na] at m/e 515.0801. The data of 3 agree with the reported data of quercetin-3-O-β-d-glucuronide-6″-methyl ester (Ryu et al., 2016).

A validated method was developed for the quantification of 1 in both ME and WE using a C₁₈ column and gradient elution system of mobile phase consisted of ultrapure water (A) acidified with 0.1 % acetic acid and Acetonitrile (B), under flow adjusted to 1.0 mL/min. The isolated pure 1 used as standard. The quantification was linear in the range of 5–1000 µg/mL (Fig. S38-S39). The correlation coefficient (R²) was 0.9993 indicating a strong correlation between concentrations and peak areas. The obtain regression equation was y = 0.0615x-0.2813 (Table S4). The method was validated as required by the ICH guide lines for accuracy, precision and robustness (Tables S5-S7) ICH, 2005). The amount of 1 in both ME and WE were determined using the developed methods (Fig. 8). The ME contains 2.99 % of 1 while WE contains 1.84 % representing more than half amount in the ME. Comparison of these values with the TPC indicated that ME contains more than 3-fold of TPC compared with WE. TP apparently plays crucial role in the activity.

Fig. 8.

HPLC chromatogram of of (-)-Epi-catechin (1), methanol extract (ME) and water extract (WE).

AA induced colitis in animal’s cause’s imbalance between oxidant and antioxidant substances (Keshavarzian et al., 1992, Dröge, 2002). It has been scientifically established that infiltration of neutrophils initiates the production of superoxide anion and starts a cascade for the production of different reactive species. This is likely resulted in generation of hydroxyl radicals and peroxides that plays a significant role in the progression of tissue necrosis and mucosal dysfunction (Closa and Folch-Puy, 2004, Grisham and Granger, 1988). Previous phytochemical screening reported the identification of several phytochemicals in the methanol extract of C. comosum such as phenolic compounds (Cheruth et al, 2016). Coumaric acid, 4-ethoxy-1,2-benzendiol and tamgermanetin previously reported from the plant along with others are expected to participate in the phrmacological effect (Belaabed et al., 2017). Our finding correlated the antioxidant power to the total TPC. The main isolated compound (-) epi-catechin is known for its antioxidant potential (Jug et al., 2021) that may explain its beneficial effects in cancer, inflammation, diabetes, and neurodegeneration managements (Shay et al, 2015). The effects observed may be caused by the combined activities of many phytoconstituents, including those mentioned herein and other compounds that have not yet been identified. Further studies are necessary to fully characterize the active compounds in C. comosum, that are responsible for these effects. Accordingly, epi-catechin and other phenolic contents are crucial for the protective effect of the ME.

4. Conclusion

Comparative in vitro and in vivo study between the ME and WE of C. comosum indicated that the ME is superior in all the conducted experiments. In the anti-ulcerative colitis study induced by AA treatment with the ME resulted in improvements in colon W/L ratio and all signs of inflammation when compared to the CC. Extensive histopathological examination showed that treatment with ME preserved the functional cytoarchitecture of the colonic mucosa and inhibited inflammatory cell infiltration, ulceration, erosions, congestion, necrosis and hyperplasia caused by AA in rats. Chromatographic purifications were conducted and enable the identification of the active components of the ME as (-)-epi-catechin. HPLC quantification indicated that the amount of (-)-epi-catechin in ME is almost double the amount in WE. TPC in ME is more than 3-fold of that in the WE. These results indicated that not only (-)-epi-catechin is responsible for the anti-ulcerative colitis but also TP plays a crucial role in the activity.

CRediT authorship contribution statement

Hassan N. Althurwi: Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing – original draft, Funding acquisition, Visualization. Mohammad Ayman A. Salkini: Software, Validation. Gamal A. Soliman: Conceptualization, Investigation, Project administration, Writing – review & editing, Data curation, Supervision. Khalid M. Alharthy: Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Abubaker Hamad: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Maged S. Abdel-Kader: Conceptualization, Formal analysis, Investigation, Investigation, Resources, Data curation, Funding acquisition, Supervision, Project administration, Visualization, Writing – review & editing, Writing – original draft.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: