Abstract
The aim of the present study was to evaluate both prophylactic and curative anti-ulcerative colitis activity and the possible mechanism of action of seven desert plant extracts. Seven desert plants from different families; Conyza dioscoridis (L.) Desf. (Asteraceae), Euphorbia hirta L. (Euphorpiaceae), Origanum syriacum L. and Salvia lanigera L. (Lamiaceae), Sisymbrium irio L., Solanum nigrum Linn. (Solanaceae) and Solenostemma arghel (Del.) Hayne. (Asclepiadaceae) were separately evaluated at three doses (125, 250, and 500 mg/kg) using the acetic acid-induced colitis model. The investigated extracts possessed prophylactic and curative anti-ulcerative colitis activities in a dose-dependent manner, where Salvia lanigera (87.9) and Solenostemma arghel (89.2) were the most effective extracts whereas the dexamesathone produced 68%. These extracts were further investigated for estimation of their mechanism of action. The in vitro potential radical (DPPH) scavenging activities of the investigated extracts were well supported with the reduction of colonic MDA content for both extracts. Suppression of the inflammatory mediator TNF-α and inhibition of both PLA2 and protease enzymes may play an important role in the anti-ulcerative colitis activities. The investigated extracts were safe for use up to 5 g/kg and the total alcohol extracts of Salvia lanigera and Solenostemma arghel (400 mg/kg for 35 d) showed no alteration on liver and kidney functions. Phytochemical screening of the investigated extracts revealed the presence of flavonoids, tannins, unsaturated sterols, and proteins which could be responsible for the activities.
Introduction
Ulcerative colitis (UC) is an inflammatory bowel disease that primarily affects the colonic mucosa and sub-mucosa. The most common symptoms of UC are ulcers and inflammation of the inner lining of the colon that lead to symptoms of bloody diarrhea, passage of pus, mucus, and abdominal cramping during bowel movements. Currently, there is no effective therapy to cure the disease but the mainstream treatment depends on the reduction of the symptoms. The treatment depends on the severity of the disease; therefore treatment is adjusted for each individualCitation1.
Inflammatory mediators contribute to the inflammatory cascade in modulating the immune system of UCCitation2. In the colonic mucosa of patients with UC, the mucosal injury and repair are repeated indicating persistent active inflammation. Recent studies have suggested that mast cells may play a role in the pathogenesis of UC. The tissues of patients with UC reveal an increase in mast cell number and accumulation of the mast cell-derived mediators, such as histamine, proteases, leukotrienes, and prostaglandin. Mast cell-derived mediators promote the recruitment of inflammatory cells in the lamina propria, suggesting that they may closely be involved in the pathogenesis of the intestinal lesion in UCCitation3.
Of the various kinds of inflammatory mediators, tumor necrosis factor-alpha (TNF-α), which is synthesized and secreted from macrophages, lymphocytes, and polymorphonuclear neutrophils, is regarded as the most prominent “first-line” cytokinesCitation4. In addition, group II phospholipase A2 was involved in the pathogenesis of intestinal inflammation as it is increased in both serum and colonic mucosa of patients with UCCitation5.
Growing evidence demonstrated the significance of oxidative stress both in the clinical and experimental studies of UC. Excessive production of reactive oxygen species (ROS) in mucosal cells induced inflammatory and immune responses which could directly or indirectly cause damage of intestinal epithelial cells, subsequently influence mucosal integrity or initiate an inflammatory signaling cascade and lead to severe impairment in experimental colitisCitation6.
The investigated extracts were reported to possess variable pharmacological properties. Anti-inflammatory and analgesic activities were reported for Conyza dioscoridis, Origanum syriacumCitation7, Euphorbia hirtaCitation8, Solanum nigrumCitation9, and Solenostemma arghelCitation10. Five extracts were potential antioxidants: C. dioscoridis, Solanum nigrum, Salvia lanigera, O. syriacumCitation6, and Solenostemma arghelCitation10.
Both C. dioscoridis and E. hirta extracts showed anti-diarrhealCitation11,Citation12, hypoglycemicCitation13,Citation14, and antimicrobial activitiesCitation15,Citation16. In addition, Salvia lanigera aerial parts ether soluble fraction was found to be highly effective against Gram-positive bacteria (Sreptomyces viridochromogenes)Citation17. Solanum nigrum extract was a potent antiulcerogenic agent with H+/K+ATPase inhibitory activityCitation18 in addition to its hepatoprotective potentialCitation19.
The aim of the present study was to evaluate the anti-UC activities of the selected plant extracts that were reported in folk medicine to possess anti-inflammatory potentials.
Results
According to the results of phytochemical screening tests, in this study, the investigated extracts contain carbohydrates, flavonoids, tannins, unsaturated sterols, proteins, and lactones. Alkaloids were only detected in C. dioscoridis and Solanum nigrum.
Flavonoids and terpenoids were the main active constituents in all the tested extracts. They were previously reported in C. dioscoridisCitation20, E. hirtaCitation21, O. syriacumCitation22, Salvia lanigeraCitation17, Sisymbrium irioCitation23, Solanum nigrumCitation24, and Solenostemma arghelCitation25.
Acute toxicity (median lethal dose; LD50) test
The tested extracts were characterized by a low degree of toxicity. The obtained results indicated that different doses of the investigated alcohol extracts (500, 1000, 2000, and 5000 mg/kg) did not produce any symptoms of acute toxicity and none of the mice died during 24 h of observation. It was suggested that oral LD50 of was higher than 5000 mg/kg. Since substances possessing LD50 higher than 50 mg/kg are non-toxicCitation26, the tested extracts were considered safe.
Effect on UC
All the investigated extracts possessed a dose-dependent anti-ulcerative colitis potentials. In both models, they reduced different parameters of UC ( and ). Only Salvia lanigera was more effective than dexamesathone (0.1 mg/kg) at two doses (250 and 500 mg/kg) and was as effective as dexamesathone at the lower dose (125 mg/kg) at both models (prophylactic and curative). All other plants at three tested doses in the curative model were less effective than dexamesathone ().
Figure 1. Curative effect of investigated plant extracts on acetic acid-induced colitis in rats. The figure shows % protection of control colitis for 22 groups of animals (n = 6), treated with alcohol extract of investigated plant extracts (125, 250, and 500 mg/kg) and dexamesathone (0.1 mg/kg) for 5 successive days after ulcerative colitis induction. The colitis was induced by slowly infusion of 2 ml (4%, v/v) acetic acid in saline into the colon through the catheter. *Significantly different from control colitis at p < 0.05. @Significantly different from dexamesathone at p < 0.05.
Table 1. Prophylactic effect of the investigated extracts on acetic acid-induced colitis in rats.
Table 2. Curative effect of the investigated extracts on acetic acid-induced colitis in rats.
In the prophylactic model, three plants (E. hirta, Sisymbrium irio, and Solenostemma arghel) were more effective than dexamesathone at two doses (250 and 500 mg/kg) and were less effective than dexamesathone at the lower dose (125 mg/kg). Extract of C. dioscoridis was significantly more effective than dexamesathone at the high dose (500 mg/kg) and as effective as it is in the other doses (125 and 250 mg/kg). In contrast, O. syriacum was significantly more effective than dexamesathone at the high dose (500 mg/kg) and as effective as it is in the dose (250 mg/kg) and significantly less effective than dexamesathone at the lower dose (125 mg/kg). Only Solanum nigrum was significantly less effective than dexamesathone at three tested doses ().
Figure 2. Prophylactic effect of investigated plant extracts on acetic acid-induced colitis in rats. The figure shows % protection of control colitis for 22 groups of animals (n = 6), pretreated with alcohol extract of investigated plant extracts (125, 250, and 500 mg/kg) and dexamesathone (0.1 mg/kg) for 5 successive days before ulcerative colitis induction. The colitis was induced by slow infusion of 2 ml (4%, v/v) acetic acid in saline into the colon through the catheter. *Significantly different from control colitis at p < 0.05. @Significantly different from dexamesathone at p < 0.05.
In both models, the most effective extract was Salvia lanigera, it produced 87.9% inhibition as compared with control colitis at the high dose, followed by Solenostemma arghel that produced 89.2%, whereas the dexamesathone produced 68%. The lowest activity from the investigated plants was reported for Solanum nigrum (43.8%) ( and ).
In vitro antioxidant activity as 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity
All the investigated extracts at concentrations of 2, 4, 6, 8, and 10 mg/ml showed potential scavenging activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Extracts of C. dioscoridis and Solenostemma arghel at the highest concentration 10 mg/ml showed the highest scavenging activity, while Solanum nigrum showed the least activity ().
Table 3. Effects of the alcohol extracts of the investigated plants on scavenging DPPH radical.
Effect on colonic thiobarbituric acid reactive substance (malondialdehyde) content
The current results approved that both Salvia lanigera and Solenostemma arghel at doses 250 and 500 mg/kg produced a significant decrease in the colonic malondialdehyde (MDA) content of acetic acid-induced colitis rats. By comparing the percent protection of control colitis, Salvia lanigera was more effective than both Solenostemma arghel and standard dexamesathone ().
Table 4. Effect of Salvia lanigera L. and Solenostemma arghel (Del.) Hayne. on colonic thiobarbituric acid reactive substance (MDA) content on acetic acid-induced ulcerative colitis in rats.
Effect on plasma TNF-α
In the present study, it has been found that the expression of TNF-α significantly increased in the acetic acid model control group and significantly reduced in the treated groups in a dose-dependent manner. Both Salvia lanigera and Solenostemma arghel (250 and 500 mg/kg) produced a significant decrease in the plasma level of TNF-α of acetic acid-induced colitis rats ().
Table 5. Effect of Salvia lanigera L. and Solenostemma arghel (Del.) Hayne. on plasma TNF-α (Tumor Necrosis Factor-alpha) level on acetic acid-induced ulcerative colitis rats.
Evaluation of phospho lipase A2 inhibitory effect
Among the seven investigated extracts, three of them Salvia lanigera, Solenostemma arghel, and C. dioscoridis showed significant results ( and ). It is worth noticing that these extracts showed the most promising results in inhibiting the catalytic activity of the pro-inflammatory hG-IIA. However, using the same extract, no inhibition of the phospholipase A2 activity of DrG-IB was observed. These results indicate a selective inhibition of the tested extracts against these two sPLA2.
Figure 3. Inhibition of sPLA2 activity. The figure shows % inhibition of the catalytic activity of the pro-inflammatory hG-IIA and phospholipase A2 activity of DrG-IB for the seven investigated plant extracts in vitro.
Table 6. Inhibition of sPLA2 activity.
Evaluation of protease inhibitory effect
Anti-protease activity against commercial serine protease, trypsin, was tested. The investigated extracts were evaluated for recovering protease inhibitor molecules. Results presented in and showed that Solenostemma arghel, showed maximum protease inhibitor activity (75% inhibition) followed by O. syriacum and Salvia lanigera extracts (72% and 44.7% inhibition, respectively).
Figure 4. Inhibition of protease activity. The figure shows % inhibition of the protease activity for the seven investigated plant extracts in vitro.
Table 7. Inhibition of protease activity.
Sub-chronic toxicity
The non-toxic nature of the alcohol extract of Salvia lanigera and Solenostemma arghel in acute toxicity study is well supported by the results of sub-chronic toxicity study. Oral dosing of the tested extract to rats in a dose of 500 mg/kg for 35 d did not show any significant effect on the activity of ALT, AST, or levels blood urea and serum creatinine as compared with the control ().
Table 8. Effect of Salvia lanigera L. and Solenostemma arghel (Del.) Hayne. on liver and kidney functions.
Discussion
The model of acetic acid-induced colitis shares many of the histologic features of UC in human beings including mucosal edema and sub-mucosal ulcerationCitation27. In the present study; rats of the normal control group showed no abnormal changes suggesting that handling procedure had no interference with the experimental outputs. Macroscopic damage parameters of the colon of control colitis rats in both models revealed dark brown lesions, mucosal hyperemia, edema, erosion, and ulceration.
The inflammatory changes of the intestinal tract were associated with a significant increase of wet weight/length of the colon specimens as an indicator of inflammation. These inflammatory indices were significantly improved by oral dosing of dexamethasone and the investigated extracts for 5 d prior to and after ulcer induction. The present results were in agreement with the results of previous workersCitation28,Citation29.
Oxidative stress and its consequent lipid peroxidation could aggravate free radicals chain reactions, disrupt the integrity of intestinal mucosa barrier, and activate inflammatory mediators. The levels of MDA were often used as an indication of oxidative damage and as a marker for free radicals-induced lipid peroxidation. Previous data showed that the levels of MDA were decreased in UC by antioxidant and anti-inflammatory agentsCitation30. In this study, the colonic MDA content was modulated in the treated groups. Therapy with the investigated extracts resulted in a marked decrease in MDA in colon tissue in a dose-dependent manner. The present results suggested that the extracts successfully inhibited lipid peroxidation induced by acetic acid, and they provided protective effects in UC probably by the radical scavenging and antioxidant properties. This may be an important and underlying mechanism of protection against UC. In addition, the investigated extracts could exert an anti-inflammatory effect by suppressing the production of active oxygen and nitrogen species.
Inflammatory mediators, including cytokines, contribute to the inflammatory cascade in modulating the immune system of UC. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the colonic mucosa significantly increased. TNF-α stimulates and induces the production of other inflammatory mediators such as ROS, and it also activates oxidative stress-responsive genes which amplify and prolong inflammation. Moreover, TNF-α has overlapping and synergetic activities to induce the production of other cytokinesCitation31,Citation32. In the present study, it has been found that the expression of TNF-α significantly increased in the acetic acid model control group and significantly reduced in the treated groups in a dose-dependent manner.
In the present study; extracts of Salvia lanigera, Solenostemma arghel, and C. dioscoridis showed a selective inhibition against the two sPLA2, these results were in agreement with Fabia et al.Citation33 who suggested that PLA2 may play an important role in acetic acid-induced colitis and inhibition of its activity may offer an alternative mode of treatment in UC.
Phospholipase A2 (PLA2) participates in the regulation of phospholipid metabolism and biosynthesis of eicosanoids, serum levels of PLA2 are suggested to reflect the disease activity in patients with UC. Several studies showed that serum PLA2 activity and levels are significantly increased in UCCitation34,Citation35. Yamaguchi et al.Citation36 demonstrated that serum PLA2 group IIA levels in UC were closely correlated with histological disease activity. Immunohistochemical study showed the production of PLA2 IIA by the polymorphonuclear cells, macrophages, and colonic epithelial cells. The authors suggested that serum PLA2 group IIA is a good candidate for assessing disease activity in UC as one of the clinical laboratory tests.
In the present work, the investigated extracts showed different potentials for recovering protease inhibitor molecules. The extract of Solenostemma arghel, showed maximum protease inhibitor activity followed by O. syriacum and Salvia lanigera extracts. Our findings are in line with several previous reports which have established a potential link between protease and protease inhibitor levels and progression of UC. In fact, increased fecal protease activity has been observed in UC patients and was correlated with disease severity while the levels of endogenous protease inhibitors such as pancreatic secretory trypsin inhibitor and an α-proteinase inhibitor are decreasedCitation37–39. Lower protease inhibitor levels could enhance tissue destruction by inflammatory proteases in the vicinity of active colonic inflammation.
Most of the activity was derived from serine proteases of pancreatic and granulocytic cell origin and was inhibited by DFP, a potent serine protease inhibitor. Further, the amount of pancreatic secretory trypsin inhibitor (PSTI), a 56-amino acid serine protease inhibitor present in the intestinal lumen, is lower in the colonic tissue from UC and Crohn’s disease patientsCitation40. A reduction in PSTI levels could render the intestinal mucosa less resistant to proteolytic attack and more susceptible to inflammation. Interestingly, camostat mesilate, a serine protease inhibitor, induced long-term remission in two patients with refractory UC. Camostat mesilate could act by inhibiting specific inflammatory proteases, thereby decreasing tissue damageCitation41.
The serum transaminase level is most widely used as a measure of hepatic injury, due to its ease measurement and high degree of sensitivity. It is useful for the detection of early damage of hepatic tissue. Since the activity of ALT and AST is specific assayable liver enzymes, their normal levels in serum of rats treated for 35 d means that the investigated extracts are not hepatotoxic.
Urea and creatinine are the most sensitive biochemical markers employed in the diagnosis of renal damage. In kidney damage, there will be retention of urea and creatinine in the bloodCitation42, therefore, marked increase in serum urea and creatinine are indications of functional damage to the kidney. By these indicators, the alcohol extracts of Salvia lanigera and Solenostemma arghel is, therefore, not nephrotoxic in rats.
Experimental
Plant materials
The aerial parts of seven desert plants from different families (C. dioscoridis (L.) Desf. (Asteraceae), E. hirta L. (Euphorpiaceae), O. syriacum L. and Salvia lanigera L. (Lamiaceae), Sisymbrium irio L., Solanum nigrum Linn. (Solanaceae), and Solenostemma arghel (Del.) Hayne. (Asclepiadaceae)) were collected during flowering stage in 2013, from different localities of Saudi Arabian Desert.
The samples were kindly identified by Dr. Jacob T. Pandalayil (Assistant Professor of Plant Taxonomy, Botany and Microbiology Department, Faculty of science, King Saud University). A voucher specimen has been deposited in the herbarium of Faculty of Sciences, King Saud University. Plant material was air-dried in shade, reduced to fine powder, packed in tightly closed containers, and stored for phytochemical and pharmacological studies.
Animals
Swiss albino mice of both sex (26–30 g) and male Wistar rats (180–200 g) were purchased from the animal house of King Saud University, KSA. Animals were maintained under standard conditions (temperature 23 ± 1.0 °C, humidity 55 ± 10%, 12 h light/12 h dark cycle) and housed in standard polypropylene cages with wire mesh top. They were fed with a standard pellet diet with water ad libitum and were allowed to adapt to the laboratory environment for 1 week before experimentation.
Extraction
Arial parts of each plant were shade dried, reduced to fine powder, stored in dark tightly closed containers for further use. Three hundred grams of each plant were separately extracted using ethanol (95%) in a Soxhlet apparatus till complete exhaustion. The total alcohol extracts were concentrated under reduced pressure at a temperature not exceeding 35 °C to yield a dry extracts. The residue obtained for each extract was weighed. Known weight of each extract was freshly prepared by dissolved or suspended in distilled water, just before administration to animals, by the aid of few drops of tween 80.
Phytochemical screening
Powdered samples from the aerial parts of the seven investigated plants were subjected to preliminary phytochemical screening according to the published methodsCitation43.
Acute toxicity (LD50) test
Mice were fasted overnight (only provided water). Plant extracts were administered orally to the groups at the dose level of 10 mg/kg and observed for 48 h for mortality. If no mortality was observed, the procedure was repeated for further higher doses such as 100, 1000, and 5000 mg/kg body weight. Toxic symptoms such as behavioral changes, motor reflexes, and mortality were observed for 48 hCitation44,Citation45.
Effect on ulcerative colitis
Experimental design
Two sets of male Wister rats were used in this study: prophylactic and curative. In each set, 24 groups each of six animals were used. Rats of groups 1 and 2 received the vehicle (5 ml/kg) and served as normal control and control colitis groups. Group 3 administered dexamethasone (0.1 mg/kg) and served as a standard group. Rats of groups 4–24 received the total alcohol extracts of the seven investigated extracts at three doses (125, 250, and 500 mg/kg) for each extract.
All medications were administered orally once daily for 5 consecutive days. In the prophylactic model, the last dose was administered 2 h before colitis induction, while in the curative model, the first dose was administrated 1 h after colitis induction. Induction of ulcerative colitis was carried out using the acetic acid-induced colitis method described by Mascolo et al.Citation46, for the prophylactic model while the curative model was first described.
Assessment of colonic lesions
The colon specimens were weighted and wet weight/length ratio was calculated for all the rats. The specimens were examined under a dissecting microscope and the lesion scores were quantified by scoring system (0–5). Ulcer area was measured using a plane glass square. Each cell on the glass square was 1 mm2 in area and the number of cells was counted and the ulcer area was determined for each colon. Ulcer index (UI) was measured by summing the lesion score and the ulcer area for each colon specimenCitation47. The curative ratio was determined according to the formula: curative ratio = control UI − test UI/control UI × 100.
Antioxidant activity (in vitro)
The antioxidant activity was evaluated using the DPPH free radical scavenging activity method described by Sreejayan and RaoCitation48. To a methanol solution of DPPH (100 μM, 2.95 ml), 0.05 ml of test extract dissolved in methanol was added at different concentrations (2, 4, 6, 8, and 10 mg/ml). Equal amounts of methanol were added to control. The absorbance was recorded at 517 nm at regular intervals of 15 s for 5 min. Ascorbic acid (100 μM) was used as a standard.
Radical scavenging activity was obtained from the following equation:
where Ac is the absorbance of control (DPPH) and At is the absorbance of test compound (certain concentration of extract), at each time.
Biochemical studies of the most effective plants
The total alcohol extracts of Salvia lanigera and Solenostemma arghel at doses 250 and 500 mg/kg were administrated to two groups of animals (n = 6). Three other groups of animals were used, two received water orally and the third received dexamesathone at dose 0.1 mg/kg orally to serve as normal control, control colitis, and standard groups. UC was induced as described before in the prophylactic model. Two days after the induction of colitis, blood samples were collected and TNF-α was measured. Colonic segments were excised, homogenized, and malondialdehyde contents were measured.
Determination of colonic thiobarbituric acid reactive substances (TBARS) or (MDA) content
The estimation of MDA was carried out using a kit supplied by Sigma-Aldrich Chemicals, St. Louis, MO.
Determination of plasma TNF-α
It was carried out using ELISA kit supplied by Ray Biotech, Inc., Norcross, GA.
Sub-chronic toxicity
Thirty rats were randomly divided into three groups. Rats of the first group received the vehicle in a dose of 5 ml/kg and left as the normal control. Rats of the second and third groups were administered the alcohol extracts of Salvia lanigera and Solenostemma arghel (500 mg/kg). All medications were administered orally daily for 35 consecutive days. Animals were maintained under identical conditions with food and water ad libitum for the entire period with close observation. At the end of the experimental period, blood samples were collected, and sera were separated to be used for the biochemical estimations. Liver functions were evaluated by measuring the serum activity of ALT and AST. Serum concentrations of urea and creatinine were determined colormetrically as measures of kidney functionsCitation49.
Inhibition of sPLA2 activity
To evaluate the potential anti-inflammatory activity of seven extracts investigated in this study, we tested the inhibitory effects of various extracts using two secreted phospholipases: hG-IIA involved in the inflammatory process and the DrG-IB which hydrolyzes dietary phospholipids. Our main objective was to find an extract which was able to inhibit selectively the pro-inflammatory phospholipase A2 group IIA with no or minimal inhibitory effect on the digestive phospholipase A2 group IB.
The test of inhibitory activity of PLA2 was performed as described by De Aranjo and RadvanyCitation50. Briefly, the substrate consisted of 3.5 mM lecithin in a mixture of 3 mM NaTDC, 100 mM NaCl, 10 mM CaCl2, and 0.055 mM red phenol as a colorimetric indicator in 100 ml H2O. The pH of the reaction mixture was adjusted to 7.6. The human group IIA phospholipase A2 (hG-IIA) or the dromedary group IB phospholipase A2 (DrG-IB) was solubilized in 10% acetonitrile at a concentration of 0.02 and 0.002 μg/μl, respectively. A volume of 10 μl of these PLA2 solutions was incubated with 10 μl of each extract for 20 min at room temperature. Then, 1 ml of the PLA2 substrate was added, and the kinetic of hydrolysis was followed during 5 min by reading the optical density at 558 nm. The inhibition percentage was calculated by comparison with a control experiment (the absence of extract).
Protease inhibitor assay
Protease inhibitor activity was assayed according to the method of KunitzCitation51 with slight modifications against trypsin. About 1 ml aliquot of trypsin (1000 units/mg) (0.5 mg/ml prepared in 0.1 M phosphate buffer pH 7) was pre-incubated with 1 ml of a suitable dilution of the protease inhibitor at 37 °C for 15 min. To the above mixture, 2 ml of 1% casein (prepared in 0.1 M phosphate buffer) was added and incubated at 37 °C for 30 min. The reaction was terminated by the addition of 2.5 ml of 20% trichloroacetic acid (TCA) solution. The reaction mixture was transferred to a centrifuge tube and the precipitated protein was removed by centrifugation at 13 000 rpm for 15 min. The absorbance of the clear supernatant was measured at 280 nm in a UV–visible spectrophotometer against appropriate blanks. One unit of inhibitor activity was defined as the decrease by one unit of absorbance of TCA soluble casein hydrolysis product liberated by the trypsin action measured at 280 nm per minute under the assay conditions. The protease inhibitor activity was expressed in terms of percent inhibition. Appropriate blanks for the enzyme, inhibitor, and the substrate were also included in the assay along with the test.
Statistical analysis
All values were expressed as mean ± S.D. Comparisons between means were carried out using a one way ANOVA test followed by the Tukey HSD test using SPSS, version 14 (SPSS, Chicago, IL). Differences at p < 0.05 were considered statistically significant.
Conclusion
All the investigated plant extracts at three tested doses showed potent dose-dependent anti-ulcerative colitis and antioxidant activities. Both Salvia lanigera and Solenostemma arghel showed the highest activity among the investigated extracts. The antioxidant potentials of the extracts were well supported with the reduction of colonic MDA. Suppression of the inflammatory mediator TNF-α and inhibition of both PLA2 and protease enzymes may be one of the possible mechanisms of action of the investigated extracts. The total alcohol extracts of Salvia lanigera and Solenostemma arghel showed no alteration on liver and kidney functions. Flavonoids and terpenoids which were the major active constituents of the investigated extracts could be partially responsible for the anti-ulcerative colitis activity.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Acknowledgements
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for the work through the research group project NO RGP-VPP-060.
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