*4.5. Evaluation of the Biological Activities of the Mucilage Extracts*

#### 4.5.1. In Vitro Hemolytic Activity

Blood (3 mL) was collected in an anticoagulant tube from a volunteer human (male, 26 years, Blood group O+) by a Phlebotomist in Punjab University Healthcentre lab (Allama Iqbal campus, University of Punjab, Lahore, Pakistan). The sample was centrifuged at 850 rpm for 5 min. The clear supernatant was poured off followed by washing of residue pellets with 5 mL of chilled (4 ◦C) phosphate buffer saline (PBS) solution (pH 7.4). Washed cells suspension was made in 20 mL cool sterilized PBS. Cells were calculated using hemocytometer and for each assay 7.068 <sup>×</sup> <sup>10</sup><sup>8</sup> cell/mL were used. 20 µL of plant mucilage were taken in an Eppendorf tube and PBS and 0.1% TritonX-100 were used as negative and positive control respectively. 180 µL of diluted blood cell suspension were added into each tube followed by incubation at 37 ◦C. After incubation for 35 min, suspension was allowed to cool for 5 min followed by centrifugation at 1500 rpm for 5 min. 100 µL of supernatant were collected and diluted with 900 µL chilled sterile phosphate buffer saline. An aliquot of 200 µL of all these samples including positive and negative control were transferred to 96-well plate. ELISA microplate reader was used to measure the absorbance at 630 nm [53].

$$\% \text{Hemolyosis} = \frac{Abs(Sample\,\,above\,\,home)}{Abs\,\,(control\,\,above\,\,home)} \times 100$$

#### 4.5.2. Acute Toxicity Study

Acute oral toxicity of MLM and MFM was determined in Wistar albino rats according to OECD guidelines 425 [54]. Limit test was performed at 5000 mg/kg where the control group was given only distilled water. The animals were observed for death or any noxious outcome in earliest four hours after the dosing and regularly for fourteen days. During this period, parameters such as weight, physical appearance, behavioral changes, injury, illness signs and mortality were observed. On the 15th day animals were anesthetized and sacrificed with an overdose of xylazine and ketamine anaesthetic drugs and blood samples

were obtained by cardiac puncture. Animals were dissected and organs such as liver and kidney were obtained for histopathological examination.

#### 4.5.3. Anti-Inflammatory Activity

Acute anti-inflammatory activity of MLM and MFM was evaluated by carrageenaninduced paw edema in rats [55]. The rats were arbitrarily separated in four groups (*n* = 5). In the first group, the animals received a dose of 10 mL/kg of distilled water (carrageenan control); meanwhile in the second group, the animals were orally administered with diclofenac sodium in a dose of 10 mg/kg (standard group) [56]. In the third and fourth groups, the animals were orally given 500 mg/kg of MLM (MLM experimental group) and MFM (MFM experimental group), respectively. After 1 h, all the animals were injected with 0.1 mL of carrageenan in normal saline (1% (*w*/*v*)) into subplantar region of left hind paw of each rat. Thickness of paw was measured using digital vernier caliper at 0 h, 1 h, 2 h, 3 hand 4 h intervals. Edema inhibition (%) was calculated using the following formula:

$$\% \text{ inhibition of edema} = \frac{\text{T}\_{\text{c}} - \text{T}\_{\text{t}}}{\text{T}\_{\text{c}}} \times 100$$

where T<sup>c</sup> = Paw thickness of control group and T<sup>t</sup> = Paw thickness of experimental group.

#### 4.5.4. Antitussive Activity

Acute antitussive activity of MLM and MFM was evaluated by SO<sup>2</sup> induced cough model in rats [57]. Each animal served his own control due to variation in number of coughs in each individual animal. Groups were treated in the following manner where in the first group, the animals were orally given codeine phosphate in a dose of 10 mg/kg. In the second and third groups, the animals were orally given 500 mg/kg of MLM and MFM respectively. The animals were exposed to SO<sup>2</sup> for 60 s after an hour of receiving the treatment and the number of coughs was counted. The number of coughs was compared before and after the treatment to determine the reduction in cough. The percentage inhibition frequency of cough was determined using the following formula:

$$\% \text{ inhibition of frequency of coupling} = \left(\frac{\text{C}\_{\text{C}} - \text{C}\_{\text{T}}}{\text{C}\_{\text{C}}}\right) \times 100\%$$

where C<sup>C</sup> is the cough frequency in control animal and C<sup>T</sup> is the cough frequency in treated animal [57].

### 4.5.5. Antiulcer Activity

#### Study Design

The animals were arbitrarily separated into 9 groups (*n* = 6) where the first group (normal group) was orally treated with distilled water (DW) 1 mL/100g [58]. The second group was orally treated with 1 mL/100 g of ethanol (ethanol group) meanwhile from 3–5 groups, the animals received orally standard drugs which are ranitidine (50 mg/kg) [59], omeprazole (20 mg/kg) [60] and sucralfate (100 mg/kg) [61], respectively. Groups 6–7 were orally administered MLM in doses of 250 and 500 mg/kg, respectively whereas groups 8–9 were orally administered MFM in doses of 250 and 500 mg/kg, respectively.

#### Ethanol Induced Acute Gastric Ulcer

Animals were prevented from food for 24 h with unrestricted excess toward water. They were deprived of water just 2 h prior starting the experimental procedure. Animals of all groups were pretreated with drugs and extract as mentioned above. After 30 min all groups were orally administered 90% ethanol (1 mL/100 g) except normal group. Animals were euthanized with an overdose of xylazine and ketamine anaesthetic drugs after 1 h of ethanol administration [62]. Animals were dissected, removed the stomachs and incised along the larger curve. Washed with ice cold normal saline and gastric mucosa was examined [63].

Ethanol Induced Chronic Gastric Ulcer

Animals were prevented from food for 24 h with unrestricted excess toward water. They were deprived of water just 2 h prior to starting the experimental procedure. All animals were treated with drug and extract for fourteen days as mentioned above. On the first day of experiment after 30 min above treatment animals were orally administered with 90% ethanol (1 mL/100 g) except normal group. From the 2nd day 30% (*v*/*v*) ethanol was orally given to all groups for fourteen days except normal group. On 14th day, all the animals were anesthetized and sacrificed with an overdose of xylazine and ketamine anaesthetic drugs after 1 h of ethanol administration [62]. Animals were dissected, removed stomachs and incised along larger curve and washed with ice cold normal saline then gastric mucosa was examined [64].

#### Parameters of Gastric Ulcer Evaluation

#### *Macroscopic and Microscopic Evaluation*

Normal saline was used to wash the stomach and the apparent changes in inner walls of stomach were carefully examined macroscopically by using magnifying glass and microscope. The number of lesions was carefully noted and was used for the determination of ulcer index. Photos were taken using mobile camera (Oppo A57, Guangdong Oppo Mobile Telecommunications Corp., Ltd., Dongguan, Guangdong, China) [65].

#### Ulcer Scoring

Based on severity, the ulcers were given scores as previously reported by Gupta et al. [66] as follows: 0 = no ulcer; 0.5 = reddish mucosa; 1 = red spots; 1.5 = hemorrhagic streaks; 2 = deep ulcers and 3 = perforations.

#### Ulcer Index

Ulcer index can be calculated as previously described by Gul et al. using the following formula [67].

Ulcer index <sup>=</sup> (UN <sup>+</sup> US <sup>+</sup> UP) <sup>×</sup> <sup>10</sup>−<sup>1</sup>

where UN = average number of ulcers per animal; US = average of severity score; UP = percentage of animals with ulcer.

#### Ulcer Protection (%)

Ulcer protection (%) was calculated described by Gul et al. using the following formula [67].

% Protection = Ulcer index of ethanol treated group − ulcer index of treated group ulcer index of ethanol treated group <sup>×</sup> <sup>100</sup>

#### Histological Analysis

Samples of stomach walls were fixed in formalin solution (10%) for 48 h and then dehydrated by washing with ascending grades of ethanol. Samples were cleaned by xylene and embedded in paraffin wax. The rotary microtome was used to cut sections of 5–6 mm thickness followed by staining with hematoxylin and eosin. The sections were examined under a microscope for histopathological variations such as ulceration, congestion, edema, necrosis and leukocyte infiltration [68].

#### Mode of Gastro-Protective Activity

#### *Estimation of the Gastric Volume*

After opening the stomach gastric content was squeezed out in a falcon tube followed by centrifugation at 1000 rpm) for 15 min, then the supernatant was collected, and its volume was determined [69].

#### Determination of the pH Value and Total Acidity

Gastric juice pH was determined using a digital pH meter as previously described by Sen et.al [68]. A 1 mL volume of gastric juice was obtained in a vial then 2 drops of phenolphthalein were added as indicator. Then, it was titrated against 0.1 N NaOH until the end point that is the change from colorless to light pink. The volume of NaOH used was determined then the following formula was used to calculate total acidity where results were expressed in terms of clinical unit mEq/L [70].

$$\text{Total acidity} = \frac{\text{Volume of NaOH used} \times \text{Normality of NaOH used} \times 100}{0.1}$$

#### Determination of Gastric Mucin Content

The glandular portion of stomach was excised and weighed, and this portion was soaked for 2 h in 10 mL of 0.1% Alcian blue solution in 0.16 M sucrose buffered with 0.05 M sodium acetate adjusted to pH = 5. Excess uncomplexed dye was removed by rinsing with 0.25 M sucrose solution at an interval of 15 min and 45 min respectively. Dye forms a complex with mucus that was extracted with 10 mL of 0.5 M magnesium chloride solution for 2 h with consecutive shaking for one minute after 30 min interval. A 4 mL volume of this solution was shaken with an equal volume of diethyl ether. The resulting emulsion was centrifuged at 3000 rpm and the absorbance of aqueous layer was taken at 580 nm. The mucin content of the sample was determined from the standard curve, which was expressed in microgram of Alcian blue extracted per gram of wet gland tissue [71].

#### Determination of the Total Protein Content

Total protein of glandular tissues homogenate was estimated by Lowry method [72]. The tissue homogenate of glandular portion of stomach was mixed with 1 mL of 0.1 M Tris-HCl buffer (pH 7.4) by a homogenizer followed by centrifugation at 1500 rpm for 15 min at 4 ◦C. The homogenate was diluted with 0.1 M tris-HCl buffer to reach 10 mL. Bovine serum albumin (BSA) was used as standard and 0.1 N of NaOH was used as blank. A 1 mL volume of the sample was mixed with 4.5 mL of reagent I that is formed by mixing 48 mL of 2% Na2CO<sup>3</sup> in 0.1 N NaOH with 1 mL of 1% NaK Tartrate in H2O and 1 mL of 0.5% CuSO4·5 H2O in H2O followed by addition of 0.5 mL of reagent II (1 part Folin-Phenol [2 N]: 1 part water) after 10 min. The test tubes were placed in the dark for 30 min and the absorbance was taken at 750 nm. BSA standard calibration curve was used to interpret the results.

#### *4.6. Gas Chromatography Coupled with Mass Spectrometry Analysis*

Monosaccharide composition of MLM and MFM was analyzed using a modified GC-MS analytical procedure previously adopted by Xia et al. [73] depending upon trimethylsilyl dithioacetal (TMSD) derivatization. A 1 µL volume of the sample was applied to Agilent 7890A Gas Chromatography coupled to Agilent 5975C Mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with HP-5MS column using a temperature range of 80 ◦C for 0 min, 80–90 ◦C at 2.5 ◦C/min, 190–252 ◦C at 2 ◦C/min, 252–300 ◦C at 25 ◦C/min, 300–310 ◦C at 25 ◦C/min and held for 15 min. Mass spectra were recorded employing total ion chromatogram (TIC) mode and interpreted using NIST 5 software [25].

#### *4.7. Statistical Analysis*

GraphPad prism 8.4.3 was used for statistical analysis. Statistical significance difference was calculated using one way ANOVA followed by Dunnett's test. The values *p* < 0.05 \*, 0.01 \*\* and 0.001 \*\*\* were considered as statistically significant.

#### **5. Conclusions**

In conclusion, the current findings suggest that mucilage extracted from the leaves and fruit of *M. parviflora* are safe for in vivo studies and could be incorporated in many significant therapeutic applications after clinical trials. Treatment with mucilage reduced cough, inflammation and ulcers in animal models. The underlying mechanism of gastro protection is in the anti-secretory and mucus protective potential of mucilage. However, further in vivo studies are recommended to comprehensively understand its exact mechanism of action. This study clearly highlights the application of *M. parviflora* mucilage in adjuvant therapy of gastric ulcers. Further studies are in progress to isolate bioactive polysaccharides and glycoprotein from this mucilage.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15040427/s1, Figure S1: GC/MS chromatograms of neutral (A) and acidic (B) polysaccharides in *M. parviflora mucilage* obtained from the fruits (MFM).

**Author Contributions:** Conceptualization, A.M. and S.I.; methodology A.M., S.I., M.R., S.S.E. and K.A.; software, F.S.Y.; resources, A.E.A., O.K., W.Y.R. and S.S.E.; writing—original draft preparation, A.M. and S.I.; writing—review and editing, F.S.Y., S.S.E. and M.L.A.; supervision, F.S.Y., S.S.E. and M.L.A.; funding acquisition, W.Y.R., A.E.A., O.K. and S.S.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia under grant no. (G-68-166-38).

**Institutional Review Board Statement:** The animal study protocol was approved by the Punjab University College of Pharmacy, (AEC/PUCP/1094 dated 11 February 2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Acknowledgments:** This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia under grant no. (G-68-166-38). All the authors, therefore, acknowledge with thanks DSR for technical and financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

