INTRODUCTION:

Medicinal plants can serve as a rich source of bioactive metabolites with the potential for drug discovery and development¹. Plant-derived natural products are considered as green medicine and have attracted scientists around the world for many years.

Because, these plant-derived medicines are affordable, readily available and have minimal side effects. Therefore, the research interest in the phytochemical analysis of medicinal plants is increasing in order to discover therapeutically potent compounds based on various pharmacological and biological studies². Bangladesh, a subtropical country, is an excellent depot of medicinal plants which belongs to various families that are yet to be fully explored³. More than 500 species of medicinal plants are being used in the preparation of traditional medicines for the treatment of various ailments⁴. It is well established that the medicinal value of these plants is attributed to the bioactive phytochemicals such as terpenoid, flavonoid, anthocyanin, phenolic compounds which can produce a distinct physiological effect in the living system. These bioactive natural products shape the foundation of modern pharmaceutical drugs⁵. In terms of beneficial phytochemicals and the shift towards natural products in the pharmaceutical industry, the biological study on medicinal plants is predominantly as noteworthy as the research on conventional drugs.

Schleichera oleosa (Lour.) Oken, commonly known as “Kushum” in Bangladesh, is extensively available in the Indian subcontinent and Southeast Asia. In English, the plant is popular as Ceylon oak, Lac tree, or Honey tree. The plant is a deciduous, semi-evergreen, medium sized (15-32 m) tree. The tree has grey-colored bark (10-12 mm thick), yellowish flower, and oval-shaped fruits. Seeds are oily and brown⁶. The plant has been shown to be valuable as traditional medicine for pain, inflammation and dysentery⁷. The seed oil of S. oleosa, called as Kusum oil, is traditionally used as a hair tonic and also for the cure of skin diseases such as itch, acne, burns, and rheumatism⁸. Seed powder is applied to wounds and ulcers. Bark of S. oleosa has analgesic, astringent properties and was found to be beneficial in the treatment of inflamed skin, ulcers and painful menstruation⁹. Phytochemical studies led to isolation of triterpenoids, steroids, tannins, fatty acids, phenol compounds from this plant¹⁰. Fruit is used in the treatment of skin problems. Bark contains triterpenoids, taraxerone and tricadenic acid A which showed antimicrobial activities against pathogens¹¹. Pettit et al. isolated hydroxylated sterols from the bark extract of S. oleosa which displayed powerful cancer cell growth inhibitory actions¹². Recently, we reported chemical isolation of 5,7-dihydroxy-4'-methoxyflavone, stigmasterol, lupeol and betulinic acid isolated from the hexane and dichloromethane soluble fractions of a methanol extract of leaves of S. oleosa¹³. So, in extension of our aforementioned works on medicinal plants^13,14,15, we herein report the potentials In vitro and in vivo bioactivities of S. oleosa.

MATERIALS AND METHODS:

Plant Material Collection and Identification:

Leaves of S. oleosa were collected from Botanical Garden, Mirpur, Dhaka-1216, Bangladesh. The plant was identified and authenticated in Bangladesh National Herbarium, Mirpur, Dhaka-1216, Bangladesh, where a herbarium sample (DACB/63763) was deposited for future reference.

Extraction of Plant Material:

Leaves were cleaned, air-dried for a week, and pulverized to a coarse powder. About 500 g of powdered materials were macerated in methanol for few days with occasional shaking and stirring. The crude extract was prepared by filtration through a cotton plug and subsequently by Whatman filter paper number 1. The filtrate was concentrated to dryness with a rotary evaporator under reduced pressure. About 5 g of dried filtrate of crude methanol extract of S. oleosa leaf (MESOL) was subjected for modified Kupchan partitioning¹⁶ into hexane (HF), dichloromethane (DMF), and ethyl acetate (EAF) soluble fractions. These plant samples as labelled by MESOL, HF, DMF and EAF were used for the evaluation of preliminary pharmacological activities by standard methods.

Chemicals and Reagents:

Butylated hydroxytoluene (BHT), streptokinase (SK), aspirin, morphine, diclofenac sodium, loperamide and glibenclamide were collected as kind gifts from Square Pharmaceuticals Limited, Bangladesh. Chemicals and reagents such as Folin-Ciocalteu, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), vincristine sulphate, Tween 80 and formalin were of analytical reagent grade.

In vitro Pharmacological Studies:

Total Phenol Content Analysis:

The total phenol content (TPC) of the plant samples was estimated using a method developed by Skerget et al.¹⁷. Briefly, 0.5 ml of test material solution (2.0 mg/ml) was mixed with 2.5 ml of Folin-Ciocalteu reagent and 2.0 ml aqueous sodium carbonate (7.5 g/l). The mixture was then incubated at room temperature for 20 min. The absorbance was measured by using a UV-visible spectrophotometer at 760 nm. The total phenol content in the test sample was denoted as gallic acid equivalent per gram (GAE/g) of the plant extract.

Antioxidant Activity Assay:

DPPH radical scavenging assay method was employed to determine the antioxidant activity of S. oleosa¹⁸. Briefly, 2.0 ml of each plant extract in concentration from 0.977 μg/ml to 500 μg/ml was mixed with 3 ml of freshly prepared DPPH solution (20 μg/ml) in methanol. The resulting solutions were incubated for 30 min in a dark place and absorbance was measured at 517 nm using a UV-VIS spectrophotometer against the blank solution of methanol. The degree of decolorization of DPPH from purple to yellow indicated the DPPH radical scavenging efficiency of the test sample. The % inhibition of DPPH radical scavenging activity was measured by the following equation:

A_DPPH - A_sample

% Inhibition of = –––––––––––––––––––––×100%

DPPH scavenging A_DPPH

Where, A_DPPH= Absorbance of the DPPH solution in methanol and A_sample = Absorbance of the plant extract (test sample) and BHT (standard) after reaction with DPPH solution. The measurement was done triplicate for accuracy. A graph of % inhibition against the plant sample concentrations was plotted. For each sample, IC₅₀ (50% inhibition concentration) value was estimated from the graph by linear regression analysis.

Cytotoxic Activity Assay:

The cytotoxic activity of plant samples was determined by brine shrimp lethality bioassay¹⁹ using shrimps (Artemia salina Leach) nauplii as test organisms. Vincristine sulphate was utilized as a positive control in this rapid bioassay. For the assay, the plant extracts of varying concentrations (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 µg/ml) were dissolved in DMSO as solvent. The test solutions of plant extract were then added to vials containing ten live nauplii in simulated seawater, prepared by dissolving 38g NaCl into 1L water. After 24 h incubation at 25 °C, a magnifying lens was used to count the number of living and dead nauplii in each vial and, the percentage of mortality was calculated for each concentration of test materials and control. The curves of percentage of mortality vs. logarithm of the plant sample concentrations were sketched, and an approximate linear correlation was viewed. Then the LC₅₀ values (50% lethal concentrations) were calculated using Microsoft Excel 2016.

Thrombolytic Activity Assay:

The In vitro thrombolytic activity of the test samples was conducted by following the protocol established by Prasad et al.²⁰, where streptokinase (SK) was used as a standard. Accordingly, from a healthy volunteer 5 ml of venous blood was collected and scattered in sterile pre-weighed Eppendorf tube (0.5 ml per tube) followed by centrifugation at 37°C for 45 min in order to clot formation. After coagulation of blood and expelling serum cautiously, the tubes were weighed by electric balance. 100 µl of each sample and controls were added and incubated these tubes for 90 min at 37°C for thrombolysis. For preparing the positive control, a commercially available lyophilized Altepase (Streptokinase) Eppendorf tube of 15,00,000 I.U. was received from Beacon Pharmaceuticals Limited, Bangladesh. In this regard, distilled water was used as negative control. After incubation, the tubes were weighed again by removing the created fluid above the clot. The following formula was used to calculate the lysis of blood clot.

Weight of the clot after lysis

% clot lysis = ––––––––––––––––––––––––––– ×100%

Weight of the clot before lysis

Anti-inflammatory Activity:

Anti-inflammatory activity was evaluated by the hypotonic solution-induced hemolysis method²¹ using aspirin as standard. In shortly, 5 ml of fresh blood was collected by a sterile syringe from a healthy volunteer and preserved it in a tube containing di-potassium salt of EDTA as an anticoagulant. Blood was washed by isotonic 0.9% saline solution in phosphate buffer (pH 7.4) through centrifugation at 3000 g for 10 min. In the current study aimed to evaluate the anti-inflammatory potential, the test sample consisted of 0.5 ml of erythrocyte suspension added with 4.5 ml of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffer saline (pH 7.4) containing either the different plant extract (2.0 mg/ml) or standard aspirin (0.10 mg/ml). 0.5 ml of erythrocyte suspension mixed with 4.5 ml of hypotonic solution (50 mM NaCl) was used as negative control. All the reaction mixtures were incubated for 10 min at room temperature, centrifuged at 3000 g for 10 min and absorbance/optical density (OD) of supernatant was recorded at 540 nm using a UV-visible spectrophotometer. The following equation was adopted to calculate the percentage inhibition of hemolysis or membrane stabilization:

OD_Control - OD_Test

% Inhibition of hemolysis = ––––––––––––––– × 100%

OD_Control

Here, OD = Optical density of the respective sample group.

Test Animals:

For in vivo pharmacological studies, Swiss albino mice (5-6 weeks old, average 25 g body weight) of both sex were commercially purchased from International Center for Diarrheal Disease and Research, Bangladesh (icddr, b). Mice were maintained under regular laboratory conditions of 25±2°C, 60-70% humidity with 12 h light/dark cycle. The animals were free to access the icddr, b formulated food and water. Prior to experiments, they were subjected to two weeks of acclimatization under laboratory condition²². The study protocol was reviewed and approved by the ethical review committee of Faculty of Biological Sciences, University of Dhaka, Bangladesh (Ref. No. 123/Biol.Scs.).

In Vivo Pharmacological Studies:

Central Analgesic Activity:

The central analgesic activity was determined by tail immersion method^14,15. Experimental mice were divided into control group (1% Tween 80 in normal saline), positive control or standard group (Morphine 2 mg/kg s.c.), and test groups (MESOL at 200 and 400 mg/kg p.o.), containing three mice in each group. Here, pain sensation in animals was induced by the thermal stimulus through dipping the tip of the tail (last 1-2 cm) in hot water at 55±1°C. The time (second) required to withdraw the tail (tail flick response) was recorded and the control groups were utilized to compare the found results from calculated data. The experimental mice were selected based on reactivity test before 24 h of the test. All the data were observed after 30, 60 and 90 min following the plant extractives administration by oral route. The average tail immersing time was calculated for each group and expressed as mean ± SD. Then the percent (%) time elongation was calculated with respect to the control using the following formula:

T_Test -T_Control

% time elongation = ––––––––––––––––––– ×100%

T_Control

Here, T_Test is the average time of tail deflection in the test group and T_Control is the average time of tail deflection in control group.

Peripheral Analgesic Activity:

The peripheral analgesic activity of S. oleosa was evaluated by formalin-induced writhing tests^14,15. Experimental mice were divided into control group (1% Tween 80 in normal saline), positive control or standard group (diclofenac sodium 50 mg/kg p.o.), and plant samples (MESOL at 200-and 400 mg/kg p.o.), containing three mice in each group. After 45 min of oral administration of test and control groups, the writhes were induced by the subcutaneous injection of 1% (v/v) formalin (10 ml/kg s.c.) in mice. The number of writhes (abdominal constrictions) was counted for each group of mice starting from 5 min after the injection of formalin up to 10 min. The percentage inhibition of writhing was calculated by using the formula:

N_C- N_T

% inhibition of writhing = –––––––––– ×100%

N_C

Here, N_C = Mean number of writhing in the control group and N_T = Mean number of writhing in the test group.

Anti-diarrheal Activity:

The anti-diarrheal activity of MESOL (200-and 400 mg/kg) was evaluated using the method of castor oil induced diarrhea in mice described by Karmakar et al²³. Twelve Swiss albino mice were allocated into four groups as like as described in the previous experiments. Loperamide (10 mg/kg p.o.) was used as a positive control. Diarrhea was induced in every mouse by oral administration of castor oil. Then each mouse was placed in a separate cage and the floor lining was changed at every hour. The number of diarrheal feces excreted by the animals was recorded for 4 hand % reduction in diarrhea by the plant extract was calculated using the following formula to evaluate anti-diarrheal activity:

D_Control - D_Test

% reduction in diarrhea= ––––––––––––––––– ×100%

D_Control

Here, D_Control refers to the average number of diarrheal defecations in the control group and D_Testrefers to that in test group in the same duration.

Hypoglycemic Activity:

The most common and acceptable method for assessing the hypoglycemic activity of plant extractives is the oral glucose tolerance test, which was adopted to evaluate the anti-diabetic property of MESOL. The procedure of this experiment described by Devi and Ramesh²⁴ was followed during the screening of hypoglycemic activity. Similarly, twelve Swiss albino mice were categorized into four groups. Normal control (1% Tween 80 in normal saline), standard/positive control group (glibenclamide 10 mg/kg p.o.), MESOL (200-and 400 mg/kg p.o.) groups were allowed to keep fasted over night before the experiment. The preplanned four groups were treated with distilled water, glibenclamide and two concentrations of test samples, respectively by oral administration. After one hour later, 10% glucose solution (2 g/kg) was administrated orally to all groups. After 30, 60, 120 and 180 min of glucose loading, blood samples were collected from tail vein and glucose level was measured using a glucometer.

Statistical Analysis:

The statistical calculation of mean, standard deviation (SD), t-test and one-way analysis of variance (ANOVA) were calculated by utilizing Microsoft Excel version 10 to determine the significant differences among these groups, p values <0.05 were pointed out for being statistically significant.

RESULTS AND DISCUSSION:

In this current study, the different solvent extracts of S. oleosa leaves was subjected for evaluation of antioxidant, thrombolytic, anti-inflammatory, analgesic, anti-diarrheal and hypoglycemic properties, and the results of our experiments were summarized in Table 1-5 and Fig.1-4.

TPC analysis is performed to find out the amount of phenolic content in the plant samples. It is widely known that phenolic compounds are plant-derived secondary metabolites which possess diverge biological functions. Due to having electron donating capacity, phenolic compounds can serve as primary antioxidant which may be associated with the lower incidence of free radical associated ailments in human populations²⁵. Also, these compounds are reported to stimulate the production of endogenous antioxidant molecules. In this study, TPC was determined by comparing the absorbance values of the different extract solutions of S. oleosa with the standard solutions of gallic acid as standard. For TPC calculation, a calibration curve (Fig. 1a) was constructed by using the absorbance of various concentration of gallic acid solution. The equation of the curve was y = 0.0167x; R² = 0.9971; where x is the absorbance at 760 nm and y is the phenolic content expressed in gallic acid equivalent. It has been clearly sketched in Fig. 1b that the TPC of distinctive extracts of S. oleosa varied from each other parts and extended from 5.12 mg to 103.23 mg of GAE/g of plant extract. Among all the extractives, the most elevated TPC was found in the ethyl acetate fraction (103.23 mg of GAE/g of plant extract) followed by MESOL (97.07 mg of GAE/g of plant extract) and aqueous fraction (76.46 mg of GAE/g of plant extract). The present study suggests that MESOL and its solvent fractions contain a notable-amount of phenolic components which refers further research for the development of natural antioxidant.

Fig. 1: (a) Standard Calibration Curve of Gallic Acid for Determination of Total Phenolic Content (TPC) and (b) TPC in Various Plant Extracts of S. oleosa Leaf. Here, MESOL = Crude methanol extract of S. oleosa leaf, HF = Hexane fraction, DMF = dichloromethane fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction of MESOL.

It’s popularly known that DPPH radical scavenging assay method is the foremost as often as possible utilized strategy for screening the antioxidant property of the plant extracts²⁶. Therefore, after TPC analysis the plant samples were tested for screening antioxidant potential by DPPH free radical scavenging activity. As shown in Fig. 2, all the test samples exhibited significant and concentration-dependent DPPH radical quenching activity. Among all the extractives, the ethyl acetate fraction exhibited a prominent antioxidant activity characterized by an IC₅₀value of 9.46 μg/ml as compared to 0.56 μg/ml exhibited by reference standard BHT (Fig. 2). The profound antioxidant effect of ethyl acetate fraction was attributable to the occurrence of maximum amount of phenol compounds (103.23 mg of GAE/g of plant extract) in this sample (Fig. 1b). The hydroxyl groups in plant samples are accountable for enabling free radical scavenging capacity. Like previous report²⁷, our observation also established a positive correlation between the anti-oxidant property and the amount of total phenolic content present in the respective test sample in each assay. Both MESOL and its aqueous fraction exerted moderate antioxidant activity with IC₅₀ values of 23.3 and 16.45 μg/ml, respectively. MESOL showed significant inhibitory effect at diluted concentration but its scavenging capacity began to decline at concentration of 100 μg/ml and this action will ultimately remain unchanged till the concentration of 500 μg/ml (Fig. 2). With respect to these results, the plant can be categorized as a promising free radical scavenger. These observed outcomes are similar to those of previous studies where the root extracts of S. oleosa were found to exhibit significant hydroxyl radical scavenging effect in different In vitro models²⁷. Thus, S. oleosa contained antioxidant components which are appropriate to develop drugs for the prevention of human diseases related to oxidative stress.

Fig. 2: DPPH Scavenging Activity and IC₅₀ Values of the Tested Plant Samples of S. oleosa. BHT = Butylated Hydroxy Toluene

Brine shrimp lethality bioassay is a straightforward method for pilot screening of cytotoxicity of plant extracts. Due to low cost and simplicity, this assay can be considered as a guide for finding a compound possessing anti-tumor and pesticidal activities²⁸. The present study showed that the extent of lethality was directly proportional to the concentration of the extract. After 24 h of observation, all the shrimps were survived in control. Even though, maximum mortalities were observed at a concentration of 400 μg/ml and least mortality at 0.78 μg/ml concentration of plant extract. The lethal concentration, LC₅₀ values of the plant extracts were obtained by a plot of the percentage of the shrimp nauplii killed against the concentrations of the plant extracts and the best-fit line data by means of regression analysis. As shown in Table 1, the highest activity was given by MESOL (LC₅₀ = 16.79 μg/ml) followed by hexane fraction (LC₅₀ = 22.19 μg/ml), ethyl acetate fraction (LC₅₀ = 23.82 μg/ml), aqueous fraction (LC₅₀ = 45.63 μg/ml) and dichloromethane fraction (LC₅₀ = 54.50 μg/ml). Similarly, Thind et al.²⁷ also reported the In vitro cytotoxic action of root extracts of S. oleosa which were shown to inhibit the growth of cancer cell lines. The cytotoxic activity displayed by the plant samples might be the result of the presence of lethal compounds in the test materials which warrants further studies.

The thrombolytic activity of S. oleosa extracts was determined as a part of searching for cardioprotective drugs from plants sources, and the results are summarized in Table 1. The estimated % clot lysis of these plant samples was ranged from 7.58% to 28.93% where the most significant clot lysis activity was displayed by dichloromethane fraction (28.93% clot lysis), followed by hexane fraction (27.39% clot lysis). However, the thrombolytic activity of this fraction was weaker than that of the standard streptokinase (66.98%). Previously, it was reported that terpene compounds may possess significant potentials of displaying thrombolytic activity²⁹. Ghosh et al.³⁰ isolated triterpenoids including taraxerone, tricadenic acid from the methanol extract of the bark of S. oleosa. This may be considered as one of the reasons for exhibiting clot lysis activity of the tested extractives.

Table 1: Cytotoxic and Thrombolytic Activities of Various Extractives of S. oleosa Leaf

Sample code	LC₅₀ (μg/ml)	% lysis of clot
MESOL	16.79	7.58
HF	22.19	27.39
DMF	54.5	28.93
EAF	23.82	11.06
AQF	45.6	9.83
VS	0.45	--
SK	--	66.98

Here, MESOL = Crude methanol extract of S. oleosa leaf, HF = Hexane fraction, DMF = dichloromethane fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction of MESOL, VS= Vincristine sulphate, SK = Streptokinase

Erythrocyte membrane stabilization assay using hypotonic solution is a common technique to screen the anti-inflammatory effect of the plant extracts. Exposure of erythrocyte to hypotonic solution, methyl salicylate leads to the membrane rupture, subsequently the breakdown of hemoglobin. Hypotonic solution-induced hemolysis leads to the accumulation of intracellular fluid, resulting in the breakdown of RBC membrane. So, the compounds having RBC membrane stabilizing properties might be suitable as anti-inflammatory agents³¹. The MESOL showed comparatively higher percentage of inhibition of hemolysis than the reference standard aspirin (74.62% vs. 61.90%, Fig. 3). The dichloromethane, aqueous and ethyl acetate fraction of MESOL have additionally shown significant anti-inflammatory activity (58.16%, 37.49% and 28.12%, respectively) whereas the hexane fraction has exhibited negligible percentage inhibition of hemolysis (9.27%). In light of these results, S. oleosa might be considered as a promising source of bioactive natural compounds, which may suffice as very good template for discovery and design of novel anti-inflammatory lead molecules to treat several sorts of infection by inhibiting various types of cytokines^32,33.

Fig. 3: Anti-inflammatory Activity of the Tested Plant Samples of S. oleosa

Nowadays commonly used pain killer agents, for example, NSAIDs and steroids have numerous drawbacks such as serious adverse effects, high cost, etc. Therefore, herbal medicines are becoming increasingly popular in the management of pain and inflammation because they are safe, more effective and are also readily available at affordable price. Like NSAIDs, many of these natural compounds exhibit analgesic action by inhibiting the inflammatory pathways^14,15. Furthermore, they can also inhibit nuclear factor-kB (NF-kB) inflammatory pathways. The exploration for new analgesic molecules from the plant resources is mounting globally. The scientific evaluation of herbal drugs with properly designed methods has exploded in recent years. In the present study, the tail immersion method is employed to study centrally acting analgesics. The test is based on the observation that narcotic drugs selectively prolong the reaction time of the tail withdrawal reflex in mice. An increase in pain reaction time or latency period indicates the level of analgesia of drug or plant extract³⁴.

The average feces count was observed for control groups (positive control vs. negative control: 8.33 ± 1.15 vs. 2.33 ± 0.57, p = 0.002) and plant samples (MESOL at the dose of 200- and 400 mg/kg: 5.0 ± 1 and 2.66 ± 0.57, respectively). The maximum reduction was observed by the extract at 400 mg/kg dose (68.0%) while the standard drug loperamide (10 mg/kg p.o.) produced 72.0% reduction of diarrheal feces in the mice. The anti-diarrheal action of MESOL may be associated with its promising antioxidant potential as observed in this study. Moreover, the diarrhea inhibitory activity of the plant samples could be mediated by the bioactive metabolites like terpenoids, glycosides, tannins and flavonoids present in S. oleosa³⁸.

The glucose loaded diabetic mice were treated with the methanolic extract of S. oleosa and an oral glucose tolerance test was used to evaluate its hypoglycemic property. The obtained average (mean ± SD) blood glucose level (mmol/L) for control and sample groups were listed in Table 5. The effects of MESOL at 200- and 400 mg/kg dose to reduce blood glucose level were observed as follows to evaluate the hypoglycemic activity in mice. Blood glucose data were monitored closely at 30, 60, 120 and 180 min after loading of glucose solution and it can be concluded that the methanolic extract of S. oleosa has no statistically significant blood glucose lowering capacity at dose of 200- and 400 mg/kg. Medicinal plants contain a diverge classes of phytochemicals which have been reported to possess hypoglycemic effect via the stimulation of insulin release by β-cells or inhibition of glucose absorption from intestine^14,15. More biological investigations are needed to isolate the bioactive component responsible for hypoglycemic action.

CONCLUSION:

The present study demonstrated the pharmacological basis for ethnomedical uses of S. oleosa. During the biological screening, the ethyl acetate fraction of S. oleosa showed prominent activity as an antioxidant due to its maximum phenol content. The crude methanol extract of S. oleosa leaf showed substantial cytotoxicity and anti-inflammatory potential in our In vitro condition. During in vivo assay, the crude methanol extract of S. oleosa leaf (200- and 400 mg/kg) produced significant (p<0.05) and a dose-dependent analgesic and anti-diarrheal effect in mice model. Moreover, our finding has also exposed several pharmacological potentials of S. oleosa that can be advantageous for the future development of new drug molecules from plant sources. However, further scientific studies would be required toward the isolation and characterization of the active components responsible for these activities.

ACKNOWLEDGEMENT:

We are thankful to Bangladesh National Herbarium, Mirpur, Dhaka-1216, Bangladesh for plant identification.

CONFLICT OF INTEREST:

The authors declare that they have no competing interests.

REFERENCES:

1. Rangasamy P, Hansiya VP, Maheswari PU, Suman T, Geetha N. Phytochemical Analysis and Evaluation of In vitro Antioxidant and Anti-urolithiatic Potential of Various Fractions of Clitoria ternatea L. Blue Flowered Leaves. Asian Journal of Pharmaceutical Analysis. 2019; 9(2): 67-76.

2. Tiwari P, Patel RK. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of ashwagandharishta Prepared by Traditional and Modern Methods. Asian Journal of Pharmaceutical Analysis. 2013; 3(4): 147-152.

3. Rashid MA, Haque MR, Sikder MA, Chowdhury AA, Rahman MS, Hasan, C. Review on Chemistry and Bioactivities of Secondary Metabolites from Some Medicinal Plants and Microbes of Bangladesh. Bangladesh Pharmaceutical Journal. 2014; 17(1): 1-17.

4. Uddin SJ, Grice ID, Tiralongo E. Cytotoxic Effects of Bangladeshi Medicinal Plant Extracts. Evidence-Based Complementary and Alternative Medicine:eCAM. 2011; 2011: 578092.

5. Pappachen LK, Sreelakshmi KS. Phytochemical Screening and In vitro Cytotoxicity Studies of Mussaenda frondosa Linn Leaves. Research Journal of Pharmacy and Technology. 2017; 10(12): 4227-4230.

6. Thind TS, Singh R, Kaur R. Rampal G, Arora S. In vitro Antiradical Properties and Total Phenolic Contents in Methanol Extract/Fractions from Bark of Schleichera oleosa (Lour.) Oken. Medicinal Chemistry Research. 2011; 20: 254-260.

7. Meshram N, Ojha M, Singh A, Alexander A, Ajazuddin, Sharma M. Significance and Traditional Medicinal Properties of Schleichera oleosa. Asian Journal of Pharmaceutical Research. 2015; 5(1): 61-64.

8. Palanuvej C, Vipunngeun N. Fatty Acid Constituents of Schleichera oleosa (Lour) Oken Seed Oil. Journal of Health Research. 2008; 22(4): 203.

9. Mahaptma SP, Sahoo HP. An Ethano Medico Botanical study of Bolangi, Orissa, India: Native Plant Remedies Against Gynecological Diseases. Ethanobotanical Leaflets. 2008; 12: 846-850.

10. Goswami S. and Singh RP. Ayurvedic, Phytochemical and Pharmacological Review of Schleichera oleosa (Lour.) Oken: a Traditional Plant with Enormous Biological Activity. World Journal of Pharmaceutical Research. 2017; 6(10): 295-309.

11. Ghosh P, Chakraborty P, Mandal A, Rasul MG, Chakraborty M, Saha A. Triterpenoids from Schleichera oleosa of Darjeeling Foothills and Their Antimicrobial Activity. Indian Journal of Pharmaceutical Sciences. 2011; 73(2): 231-233.

12. Pettit GR, Numata A, Cragg GM, Herald DL, Takada T, Iwamoto C, Riesen R, Schmidt JM, Doubek DL, Goswami A. Isolation and Structures of schleicherastatins 1-7 and schleicheols 1 and 2 from the Teak Forest Medicinal Tree Schleichera oleosa. Journal of Natural Product. 2000; 63(1): 72-78.

13. Anjum N, Hossain MJ, Haque MR, Chowdhury A, Rashid MA, Kuddus MR. Phytochemical Investigation of Schleichera oleosa (Lour.) Oken Leaf. Bangladesh Pharmaceutical Journal. 2021; 24(1): 33-36.

14. Islam M, Jannat T, Kuddus MR, Rashid MA, Haque, MR. In vitro and in vivo Evaluation of Pharmacological Potentials of Campsis radicans L. Clinical Phytoscience. 2019; 5: 42.

15. Fahad T, Kuddus MR, Hasan C. Phytochemical and Biological Studies of Bark Extract of Miliusa velutina (Dunal) Hook. f. & Thomson. Dhaka University Journal of Pharmaceutical Sciences. 2020; 19(2): 125-131.

16. VanWagenen BC, Larsen R, Cardellina JH, Randazzo D, Lidert ZC, Swithenbank C. Ulosantion, a Potent Insecticide from the Sponge Ulosa ruetzleri. Journal of Organic Chemistry. 1993; 58(2): 335-337.

17. Skerget M, Kotnik P, Hadolin M, Hras AR, Simonic M, Knez Z. Phenols, Proanthocyanidins, Flavones and Flavonols in Some Plant Materials and Their Antioxidant Activities. Food Chemistry. 2005; 89(2): 191-198.

18. Jenifer P, Balakrishnan CP, Pillai SC. In-vitro Antioxidant Activity of Marine Red Algae Gracilaria foliifera. Asian Journal of Pharmacy and Technology 2017; 7(2): 105-108.

19. Saha D, Paul S. Cytotoxic Activity of Methanolic Extract of Alpinia conchigera Griff (Family: Zingiberaceae). Asian Journal of Pharmaceutical Research. 2012; 2(2): 86-88.

20. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ, Taori GM, Daginawala HF. Effect of Fagonia arabica (Dhamasa) on In vitro Thrombolysis. BMC Complementary Alternative Medicine. 2007; 7: 36.

21. Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN. Membrane Stabilizing Activity-a Possible Mechanism of Action for the Anti-inflammatory Activity of Cedrus deodara Wood Oil. Fitoterapia. 1999; 70(3): 251-257.

22. Hossain MJ, Sultan MZ, Rashid MA, Kuddus MR. Does Rabeprazole Sodium Alleviate the Anti-diabetic Activity of Linagliptin? Drug-Drug Interaction Analysis by In vitro and in vivo Methods. Drug Research. 2020; 70(11): 519-527.

23. Karmakar UK, Rahman KS, Biswas NN, Islam MA, Ahmed MI, Shill MC, Paul P, Kamruzzaman M. Antidiarrheal, Analgesic and Antioxidant Activities of Trapa bispinosa Roxb. Fruits. Research Journal of Pharmacy and Technology. 2011; 4(2): 294-297.

24. Devi MRC, Ramesh B. Hypoglycemic Activity of Leaves of Bougainvillea spectabilis Extract in Streptozotocin-Induced Diabetic Rats. Asian Journal of Pharmaceutical Research. 2018; 8(2): 99-103.

25. Sahu P, Sahu K, Dubey RD, Chatterjee S, Chatterjee T. Herbal Antioxidants: A Review. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(6): 427-434.

26. Sadeghi Z, Valizadeh J, Azyzian Shermeh O, Akaberi M. Antioxidant Activity and Total Phenolic Content of Boerhavia elegans (choisy) Grown in Baluchestan, Iran. Avicenna Journal of Phytomedicine. 2015; 5(1): 1-9.

27. Thind TS, Rampal G, Agrawal SK, Saxena AK, Arora S. Evaluation of Cytotoxic and Radical-Scavenging Activities of Root Extracts of Schleichera oleosa (Lour.) Oken. Natural Product Research. 2012; 26(18): 1728‐1731.

28. Oany AR, Siddikey AA, Hossain, MU. Islam R, Emran AA. A Preliminary Evaluation of Cytotoxicity, Antihyperglycemic and Antinociceptive Activity of Polygonum hydropiper L. Ethanolic Leaf Extract. Clinical Phytoscience. 2017; 2: 2.

29. Pietri S, Maurelli E, Drieu K, Culcasi M. Cardioprotective and Anti-oxidant Effects of the Terpenoid Constituents of Ginkgo biloba extract (EGb 761). Journal of Molecular and Cellular Cardiology. 1997; 29(2): 733-742.

30. Ahmed F, Rahman MS. Preliminary Assessment of Free Radical Scavenging, Thrombolytic and Membrane Stabilizing Capabilities of Organic Fractions of Callistemon citrinus (Curtis.) skeels Leaves. BMC Complementary Alternative Medicine. 2016; 16: 247.

31. Ezeja M, Omeh Y, Ezeigbo L, Ekechukwu A. Evaluation of the Analgesic Activity of the Methanolic Stem Bark Extract of Dialium guineense (Wild). Annals and Medical Health Sciences Research. 2011; 1(1): 55-62.

32. Hossain MJ, Kuddus MR, Rashid MA, Sultan MZ. Understanding and Dealing the SARS-COV-2 Infection: An Updated Concise Review. Bangladesh Pharmaceutical Journal. 2021; 24(1): 61-75.

33. Hossain MJ, Rahman SMA. Repurposing Therapeutic Agents Against SARS-CoV-2 Infection: Most Promising and Neoteric Progress. Expert Review of Anti-Infective Therapy. 2020; 23: 1-19.

34. Shakkeel MKV, Kumar A, Babu VD, Swamy NVB. Pharmacological Evaluation of Trichilia connaroides Bark for Analgesic and Anti-inflammatory Activity in Experimental Animal Models. Asian Journal of Pharmaceutical Research. 2015; 5(3): 138-144.

35. Demsie DG, Yimer EM, Berhe AH, Altaye BM, Berhe DF. Anti-nociceptive and Anti-inflammatory Activities of Crude Root Extract and Solvent Fractions of Cucumis ficifolius in Mice Model. Journal of Pain Research. 2019; 12: 1399-1409.

36. Shulan S, Wang T, Duan JA, Zhou W, Hua YQ, Tang YP, Yu L, Qian DW. Anti-inflammatory and Analgesic Activity of Different Extracts of Commiphora myrrh. Journal of Ethnopharmacology. 2011; 134: 251-258.

37. Rahman MK, Chowdhury MAU, Islam MT, Chowdhury MA, Uddin ME, Sumi, CD. Evaluation of Antidiarrheal Activity of Methanolic Extract of Maranta arundinacea Linn. Leaves. Advances in Pharmacological and Pharmaceutical Sciences. 2015; Article ID 257057.

38. Mondal M, Hossain MS, Das N. et al. Phytochemical Screening and Evaluation of Pharmacological Activity of Leaf Methanolic Extract of Colocasia affinis Schott. Clinical Phytoscience. 2019; 5: 8.

Received on 07.02.2021 Modified on 29.04.2021

Research J. Pharm. and Tech 2022; 15(1):113-121.

DOI: 10.52711/0974-360X.2022.00019

Group	Dose			Average immersion time (in sec)
		After 30 min		After 60 min		After 90 min
		mean ± SD	p-value	mean ± SD	p-value	mean ± SD	p-value
Control	10 ml/kg	1.86±0.22	-	1.99±0.16	-	2.310±0.35	-
Morphine	2 mg/kg	4.80±0.36	0.0006	9.35±0.36	<0.0001	13.85±0.13	<0.0001
MESOL	200 mg/kg	3.33±0.22	0.0011	5.42±0.55	0.0047	7.140±0.42	<0.0001
MESOL	400 mg/kg	3.98±0.19	0.0001	6.06±0.25	<0.0001	8.090±0.61	0.0003

Group	Dose	Writhing count			Writhing (mean ± SD)	p-value	% inhibition
Group	Dose	Mice-1	Mice-2	Mice-3	Writhing (mean ± SD)	p-value	% inhibition
Control	10 ml/kg	20	19	21	20.0 ± 1	-	-
Standard	50 mg/kg	6	4	5	5.0 ± 1	<0.0001	75.00
MESOL	200 mg/kg	12	8	5	8.33 ± 3.5	0.0155	58.33
MESOL	400 mg/kg	8	6	7	7.0 ± 1	<0.0001	65.00

Group	Dose	Total feces count after 4 h			Average (mean ± SD)	p-value	% Reduction of diarrhea
Group	Dose	Mice-1	Mice-2	Mice-3	Average (mean ± SD)	p-value	% Reduction of diarrhea
Control	10 ml/kg	7	9	9	8.33 ± 1.15	-	-
Loperamide	10 mg/kg	2	2	3	2.33 ± 0.57	0.0020	72.00
MESOL	200 mg/kg	6	4	5	5.00 ± 1.00	0.0097	40.00
MESOL	400 mg/kg	3	2	3	2.66 ± 0.57	0.0023	68.00

Sample	Dose	Mean blood glucose level (mmol/L)
Sample	Dose	0 min	+30 min	+60 min	+120 min	+180 min
Control	10 ml/kg	6.8±0.75	22.86±4.75	20.2±4.78	7.87±1.88	7.13±0.81
Standard	10 mg/kg	5.3±1.45^#	20.16±3.88^#	10.33±1.76^*	4.16±0.72^#	2.76±0.15^*
MESOL	200 mg/kg	4.0±0.70^*	19.97±8.22^#	11.97±1.94^*	6.53±4.65^#	4.16±2.05^*
MESOL	400 mg/kg	4.83±2.67^#	17.46±5.05^#	12.53±5.42^#	6.63±3.20^#	6.0±2.66^#