The global prevalence of diabetes has almost doubled among adults over 18 years of age in 2014 than it was in 1980 and the prevalence has been rising more rapidly in middle- and low-income countries¹. According to WHO, diabetes will reach the seventh position of leading causes of mortality by the year 2030.

At present diabetes is commonly treated with insulin and different oral hypoglycemic drugs such as biguanides, sulfonylureas, glinides etc. Though many of these drugs possess severe side effect, there is no alternative at this moment².

Therefore, it is now essential to explore safer anti-diabetic drugs with better activity. Many ayurvedic studies showed that extracts of medicinal plants could be a potential source of anti-diabetic drugs and thus are recommended in the treatment of diabetes³. Hence, the discovery of newer hypoglycemic agents from these abundant medicinal plants is indeed promising to work with.

Around 525000 child deaths below five years old occur each year in the world due to diarrheal diseases and these become the second most common cause of child mortality⁴. Many people all over the world are using different parts of various plants as an easy access to treat and cure diarrhea. A large number of literature reveals the custom of using extracts of medicinal plants for such purpose^5,6. In addition to these, commonly used sedative-hypnotic drugs to treat different psychiatric ailments such as anxiety, insomnia, depression etc. were also found to be associated with increased risk of mortality^7,8. Severe side effects allied with widespread use of these therapies include physical dependence, tolerance and loss of cognitive function^9-11. Furthermore, anxiety and depression have huge influence in our daily life by reducing our ability to work and productivity. A recent study in association with WHO assessed that loss of productivity due to anxiety and depression disorders costs 1 trillion USD of global economy each year¹². Therefore, development of new sedative-hypnotic drugs with fewer side effects would be a promising approach to combat different psychiatric disorders.

Clerodendrum viscosum Vent is a terrestrial shrub of Verbenaceae family and is extensively distributed as a weed in tropical and subtropical areas of the world. Its common name is hill glory bower and locally known as Ghentu in Bangladesh. In traditional and siddha medicine, it is one of the commonly used natural cures in various diseases. Leaf extract of Clerodendrum viscosum possesses anti-inflammatory, analgesic, antimicrobial, antidiarrheal, antidiabetic, antioxidant, antiproliferative and neuropharmacological properties^13-15. The root extracts of Clerodendrum viscosum have anthelmintic, antibacterial, anti-fungal, anti-inflammatory and analgesic properties¹⁶. However, there is no scientific report demonstrating the antidiabetic, antidiarrheal and sedative activities of Clerodendrum viscosum roots. Therefore, in the present study we investigate the antidiabetic, antidiarrheal and sedative effects of methanolic extract of Clerodendrum viscosum roots (MECV) in mice for the first time. We found that MECV possesses moderate to strong antidiabetic, antidiarrheal and sedative activities.

METHODS:

Plant material collection:

The plant Clerodendrum viscosum was collected from Gajipur, Dhaka and identified by taxonomist of National Herbarium, Bangladesh situated at Mirpur in Dhaka, Bangladesh. The roots were separated and air dried for 3-5 days followed by oven drying for 24 hours at 37˚ C. Then the roots were ground to a fine powder and stored in an airtight storage container.

Plant extract:

Two hundred fifty grams of the powdered root was weighed in a clean amber colored container (5.0 L) and 2.0 L of methanol was added. The container was sealed and the mixture was kept for 3 days in a cool and dry place with occasional stirring and shaking. The total mixture was then filtered using a cotton plug followed by filter paper (Whatman No.1). Methanol was evaporated from the filtrate by a rotary evaporator at ambient temperature and 32g of crude extract was obtained. For the ease of oral administration, a suspension of the crude extract was prepared by adding small amount of suspending agent (tween 80) to the extract and triturated in unidirectional way. Normal saline was then slowly added to the mixture to make the final volume of the formulation and the suspension was stabilized by vortex mixer.

Reagents and chemicals:

All chemicals and reagents used in the study were of analytical grade. Standard Ioperamide (LPD), glibenclamide (GBC), diazepam (DZM), phenobarbitone and tween 80 were collected from Square Pharmaceuticals Ltd., Bangladesh. Castor oil (WELL’s Heath Care, Spain) was collected from local suppliers.

Animals:

Swiss-albino mice irrespective of sex of 4-5 weeks age were collected from the International Centre for Diarrhoeal Diseases and Research, Bangladesh (icddr, b). The mice were accustomed with the test environment prior to the experiment since they are very sensitive to the changes in environment. For this the animals were kept in standard polypropylene cages in a 12 h light-dark cycle under controlled room temperature for at least 7 days (24±2ºC; relative humidity 60-70%) and icddr, b formulated food for rodent was fed during this period.

For each experiment, four (4) groups of animals were used consisting of three (3) mice per group and the groups were marked as group I, II, III, and IV. Each group was treated individually: group I treated as negative control, Group II received reference drug of each experiment, Group III and IV received the test materials. The mice were weighed accurately before any treatment to adjust the doses of the test samples and control materials. To observe the response of each mice individually in a group, the animals were marked as 1, 2, and 3.

Acute oral toxicity study:

MECV was subjected to the acute oral toxicity study according to the guidelines of acute oral toxicity test 423 set by Organization for Economic Cooperation and Development (OECD)¹⁷. Since the experiment revealed that the administration of MECV was safe for the test animals up to a dose of 2000mg/kg body weight, a dose of 200mg/kg (MECV 1) and 400mg/kg (MECV 2) body weight were chosen as experimental doses in the current study.

Determination of antidiabetic activity:

The oral glucose tolerance test (OGTT)¹⁸ was carried out in healthy mice after fasting overnight (18 hours). Blood glucose level of each animal of each group was determined at zero hour and 0.6ml 10% glucose solution (2gm/kg body weight) was administered orally. After 25 minutes, extracts MECV 1 and MECV 2 were administered into the test groups (group III and group IV). Glibenclamide (GBC) (10mg/kg body weight) solution and saline water were used as positive and negative controls, respectively. After 60, 120 and 180 min of glucose loading, glucose levels were measured in tail vein blood by using glucometer.

Determination of anti-diarrheal activity:

The anti-diarrheal activity of MECV was assessed following the method of castor oil induced diarrhea in mice¹⁹. Group I (negative control) received 1% tween 80 in normal saline at dose 10ml/kg orally. Group II (positive control) received LPD at the dose of 50mg/kg orally. Group III and IV (test groups) received MECV 1 and MECV 2, respectively. After 60 min of administration, each mouse was fed with 1ml of highly pure analytical grade of castor oil to induce diarrhea. To observe the response, each mouse was placed in an individual cage with a clean floor lining. During the observation period of 4 hours the floor lining was changed at every hour and the frequency of diarrhea and delay of defecation time was recorded. The results of the test groups were compared against that of the control group to estimate the anti-diarrheal activity of MECV.

Determination of sedative activity:

Phenobarbitone induced sleeping time test was performed according to the method of Muhammed et al., 2013²⁰. Group I was the control group which received normal saline water containing 1% Tween 80 solution. Group II received DZM as the standard drug. The experimental groups (group III and IV) were administered with MECV 1 and MECV 2, respectively. After thirty minutes phenobarbitone sodium (25mg/kg body weight) was administered (i.p.) to each animal in each group. The onset of sleep and total sleeping time were recorded for both the control groups and MECV treated groups.

Ethical clearance:

Ethical clearance was provided by the institutional Biosafety, Biosecurity and Ethical Clearance Committee of the institute where the research was conducted.

Statistical analysis:

In each experiment the results obtained for MECV treated groups were compared with that of the control group. The results are presented as mean±standard deviation (SD). The Stuent’s t-test was used to analyze. P<0.05-0.001 were considered as statistically significant.

RESULTS:

Acute toxicity study:

The preliminary acute oral toxicity study revealed that MECV is nontoxic up to a dose of 2000mg/kg body weight since the extract at this dose did not cause any major changes in the physiology or death of the experimental animals.

Oral glucose tolerance test (OGTT):

Blood glucose level of the negative control mice, positive control mice treated with GBC and different doses of the extract MECV 1 and MECV 2 at 0, 60, 120 and 180 min after oral administration of glucose (2g/kg) is displayed in Figure 1. In the negative control group, blood glucose level was increased after 60 min which maintained the level over the next 120 min. MECV 1 and MECV 2 significantly reduced (P < 0.05) the blood glucose level at 60 min when compared with the control mice and continued this low level over the next 120 min. After 3 hours, we figured out a balanced ratio between standard and test groups which also showed significant value.

Figure 1: Effect of MECV on oral glucose tolerance test in mice. Values are expressed as mean ± SD (n = 3); *p < 0.05 when compared with positive control (Student`s t-test).

Anti-diarrheal activity:

In the screening of anti-diarrheal activity using castor oil induced diarrhea method, MECV showed statistically significant anti-diarrheal activity (Table 1). All the three groups namely group II (positive control) which received LPD, group III (MECV 1) and IV (MECV 2) had significant (p<0.05) anti-diarrheal activity when compared with the control. MECV 1 and MECV 2 decreased the total number of wet feces formed due to castor oil (4.33±0.94 and 4.00±0.81) in comparison to the positive control (3.67±0.47).

Sedative activity:

To assess the sedative activity of MECV, total sleeping time was determined in phenobarbitone induced mice. MECV 1 and MECV 2 showed statistically significant sedative activity (p<0.05) with respect to the control suggesting that the extract potentiated the phenobarbitone induced sleeping time (Figure 2). However, no relation was observed between the duration of sleeping time and dose in the present study.

Figure 2: Determination of sleeping time in mice. Values were expressed as mean ± SD (n = 3); *p < 0.05 when compared with the positive control (Student`s t-test).

DISCUSSION:

Traditional medicine is still highly relevant in people of many developing countries fulfilling their primary health care demands. In addition to it, 40 percent of the population in western nations use traditional medicine for mitigation of diseases²¹. However, efficacy of these plant derived traditional medicines are still needed to be proved scientifically. Traditionally, a number of species of Clerodendrum genus are in use as medicine by various tribes in many countries like China, India, Japan, Korea and Thailand. Leaf and root extracts of other species of Clerodendrum such as C. indicum, C. phlomidis, C. serratum, C. trichotomum, C. chinense and C. petasites were used to treat inflammatory diseases: asthma, rheumatism, ophthalmia, incephalagia etc.^22-26. Antidiabetic, antihypertensive and sedative properties have been reported for C. phlomidis, C. colebrookianum, C. calamitosum and C. trichotomum^23,25,27-28. The experimental models in this study were therefore employed to validate antidiabetic, antidiarrheal and sedative efficacy of MECV.

Phytochemical screening of MECV in the present study revealed the presence of alkaloids, glycosides, phenols, tannins, flavonoids, coumarins and steroids. Hypoglycemic action of MECV was evaluated by oral glucose tolerance test. GBC was used as standard in this study which reduces blood sugar level by improving insulin secretion from beta cells of pancreas thus providing long term effect. MECV showed statistically significant (p<0.05) blood glucose lowering activity at the doses of 200 and 400mg/kg body weight, which was comparable to the standard drug, GBC. The result was dose-dependent and confirmed the potential of the extract in the control of glucose level as observed in oral glucose tolerance test. Literature search showed that flavonoids, phenols and coumarines exhibit hypoglycemic activity²⁹. MECV was also found to contain these compounds which might be responsible for the anti-diarrheal activity. Different species of clerodendrum showed antidiabetic activity. Ethanol extract of Clerodendrum phlomoidis L leaves³⁰ and methanolic extract and dichloromethane fraction of Clerodendrum volubile leaves were reported to exhibit significant antidabetic activity³¹.

Antidiarrheal activity was examined in mice by using the method of castor oil induced diarrhea where MECV was used to determine the amount of reduction of diarrheal feces. MECV showed the reduction of diarrheal feces in mice by 51.85% and 65.56% at 200 and 400mg/kg doses. LPD (Ioperamide HCl) was used as positive control or standard in the present study. Castor oil contains ricinoleic acid (85-95%) which has laxative property. After hydrolysis in the small intestine castor oil releases ricinoleic acid which exerts its laxative effect by irritating the intestinal mucosa, and ultimately diarrhea occurs. In addition to this condition, prostaglandins stimulate gastrointestinal motility and secrete water and electrolytes. In our study, the extract produced 51.85% and 65.56% reduction of diarrhea at doses of 200mg/kg body weight (MECV 1) and 400mg/kg body weight (MECV 2), respectively. This result was comparable to the standard drug LPD which presented 59.26% reduction of diarrhea at the dose of 50mg/kg body weight. It is also well established that LPD inhibits diarrhea induced by castor oil⁵. Thus the result suggested that MECV contains antidiarrheal components. It was found that flavonoids, alkaloids, tannins, saponins, and steroids are usually responsible for the antidiarrheal activity of medicinal plant extracts³². We hypothesize that since our extract was crude in nature, there is a huge possibility that flavonoids and phenols within the methanolic extract was responsible for the anti-diarrheal activity. Previous studies of other species of Clerodendrum also confirmed their antidiarreheal property. Pal et al., 2012 showed that chloroform fraction and methanolic concentrate of Clerodendrum indicum leaf at a dose of 400mg/kg body weight exhibited inhibition of defecation at 21.74% and 26.96% in comparison to the standard group (37.39%)³³. Significant anti-diarrheal activity was noticed for the methanolic leaf extract of Clerodendrum infortunatum compared with the standard and control group¹⁴.

MECV showed moderate sedative activity in comparison to the control in phenobarbitone induced sleep method. In this trial of phenobarbitone initiated sleep in mice, the potentiated impact of MECV in mice was observed. The extract increased the duration of sleeping time in experimental animals. Phytochemicals, for example flavonoids, terpenes and saponins have been found to have sedative impact³⁴. The observed sedative effect of MECV was similar to diazepam, the standard drug used in the study. Benzodiazepines exert their sedative actions through interaction with the gamma aminobutyric acid (GABA) [20]. It could be acclaimed that flavonoids, terpenes or saponins of the Clerodendrum viscosum add to the sedative impact of this plant through central benzodiazepine receptors since the overall effects of MECV was comparable to diazepam.

CONCLUSION:

The findings of the current study suggest that the methanolic extract of Clerodendrum viscosum root may be a promising source for the exploration of new antidiabetic and antidiarrheal drugs. The study also revealed that the extract acts as sedative agent by potentiating the phenobarbitone induced sleeping time. Further research can be carried out to isolate the chemical constituents of the extract responsible for the therapeutic activities observed. Further in vitro and in vivo studies may also be performed to investigate and standardize the effects.

ACKNOWLEDGEMENT:

Authors acknowledge Ms. Kaniz Taskina for helping during the sedative activity test performance of the extract. Authors confirm that the project has no funding or grants.

CONFLICT OF INTEREST:

The authors proclaim that they have no conﬂicts of interest.

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Received on 25.01.2019 Modified on 27.02.2021

Research J. Pharm. and Tech. 2021; 14(6):3307-3312.

DOI: 10.52711/0974-360X.2021.00575

Group (n=3)	Treatment	Total number of feces	Inhibition of defecation (%)	Total number of diarrheal feces	Inhibition of diarrhea (%)
I	Castor oil + saline	10.67 ± 1.24	—	9.00 ± 0.82	—
II	Castor oil + LPD	4.00 ± 0.82*	62.50	3.67 ± 0.47*	59.26
III	Castor oil + MECV 1	5.00 ± 1.41*	53.10	4.33 ± 0.94*	51.85
IV	Castor oil + MECV 2	4.33 ± 0.47*	59.38	4.00 ± 0.81*	55.56