INTRODUCTION:

Medicinal plants have always been a fundamental segment of healthcare system since times of antiquity. The medicinal property of any plant is contributed through some potential chemical constituents that have the capability to produce some sort of effects on the physiological processes of the body. Diabetes mellitus is a graving health condition that affects people with all socio-economic status and is characterized by continual accumulation of glucose in blood leading to excessive urination, weight loss, fatigue and stupor.¹

It is the most common endocrine disorder and it is estimated that by the end of 2025 over 300 million people worldwide would be affected by diabetes mellitus.² The use of ethnobotanicals has been known to have a long standing record in the treatment of abnormalities of blood glucose.^3-13 India has been known to have a rich source of ethnomedicine available in the various systems of alternative medicine practiced in the country.

The leaves of Crinum asiaticum have been used to treat inﬂammation and skin diseases. It has also been reported to have antibacterial activity and is used in treatment of ulcers. The seeds of Crinum asiaticum are used as purgative and the stem is used in the treatment of Gonorrhoea.¹⁴

The aim of the present study was to investigate the glucose lowering potential of C. asiaticum by inducing diabetes in rats by alloxan.

MATERIAL AND METHODS:

Plant material:

C. asiaticum leaves were procured from the local area of Prayagraj, Uttar Pradesh, India during the month of May and the plant was taxonomically authenticated form Botanical Survey of India. The shade dried leaves were ground to powder prior to extraction with solvents of varying polarity.

Extraction of leaves of C. asiaticum:

The freshly collected leaves of C. asiaticum were shade dried, powdered and extracted successively using petroleum ether, chloroform, ethyl acetate, ethanol and water by cold maceration technique. The progressive dissolvable extracts were concentrated by rotary evaporator under vacuum and at low temperatures. The yield of the extracts was recorded as percentage of the dried leaf powder.

Preliminary screening of the leaf powder and extracts¹⁵:

The dried leaf powder was subjected to determination of total-ash value, acid-insoluble ash, water soluble debris, alcohol soluble extractive, water soluble extractives and loss on drying. The extracts obtained from various solvents were screened for the presence of phtyochemicals like carbohydrates, proteins, gums, mucilage, aminoacids, steroids, glycosides, alkaloids, tannins and phenolics.

Detection of carbohydrates:

To 2ml of the extract, 5 to 8 drops of Fehling's solution (boiling) was added. Formation of a birck-red colored) precipitate was an indicator for the existence of reducing sugar.

Detection of proteins:

0.25% w/v ninhydrin reagent was mixed dropwise to the extracts and the mixture was boiled for some duration. Development of a blue-color in the solution was indicative of the presence of amino acids.

Detections of terpenoids (Salkowski’s test):

5ml extract was dissolved in chloroform (2ml) and followed by the addition of 3ml conc. sulphuric acid. Development of a reddish-brown hue at the joint of the tow liquids indicated the existence of terpenoids in the extract.

Testing of steroids (Liebermann-Burchard test):

1ml plant extract was taken in a test tube and solubilized using 10ml chloroform; 1ml of acetic anhydride was added to the solution. 2 drops of conc. sulphuric acid was flown down the sides of the test tube. If the color of the solution turned red to blue and finally greenish, it exhibited the presence of steroids.

Testing for Cardiac glycosides (Keller Killiani test):

1ml of leaf extract was solubilized in 1ml glacial acetic acid by gentle heat and the solution was allowed to cool followed by the addition of 2 to 3 drops of ferric chloride. To this solution, 2ml of conc. H₂SO₄ was carefully added along the sides of the test tube. Development of reddish-brown colored ring at the junction of two layers denotes the existence of glycosides.

Testing for flavonoids:

To 2ml of the extract, 5ml ethanol (95%), few drops of concentrated HCl and 0.5g magnesium turnings were added. Appearance of orange, red to purple or pink color indicated the presence of flavonols.

Testing for alkaloids:

The extract as dissolved in dilute HCl, filtered and the following tests were carried out to confirm alkaloids.

Mayers test:

To 2ml of the filtrate, Mayer’s reagent (1-2 drops) was added by the side of the test-tube. Occurrence of white or creamy precipitate denotes positive test for alkaloids.

Wagners test:

To 1ml of the filtrate, few drops of Wagner’s reagent were mixed alongside the walls of the test tube. If a reddish-brown precipitate occurred, the test was considered as positive for alkaloids.

Testing for phenolics (Ferric Chloride Test):

1ml extract was solubilized in 2ml of distilled water followed by the addition of few drops of 10% ferric chloride solution. Formation of dark green color denotes the existence of Phenolic compounds.

Detection of tannins (Ferric Chloride Test):

1ml of extract was solubilized in 10ml distilled water and filtered. Aqueous Iron (III) chloride (1%w/v) solution was added to the filtrate. The occurence of intense green, purple, black or blue color in the solution denotes for the presence of tannins.

Animals:

Grown-up albino wistar rats (180-200g) of either sex were housed in polypropylene cages under standard conditions (12h light; 12 h dark cycle; 23±2°C, 50±5% relative humidity). The animals were fed with standard pellet diet and access to water ad libitum. All the animal experiments were planned and performed in accordance with the guidelines approved by CPCSEA, and Institutional Animal Ethics Committee (SIP-IAEC-01-15-01).

Acute toxicity Study:

The animals were fasted overnight and water ad libitum, followed by the oral administration of the extracts at dose of 5, 50, 300 and 2000mg/kg and observation for mortality over 14 days. The same dose was re-administered to confirm the toxic dose.

Antidiabetic activity:

Induction of experimental diabetes:

For induction of diabetes, animals were subjected to overnight fast (free access to water) for 12 hours to make them additionally susceptible to developing diabetes¹⁶. Diabetes was induced in the test animals by intraperitoneally administrating alloxan monohydrate (150mg/kg body weight) solubilized in normal saline. After 72 h mice with blood glucose range of 200 to 350 mg/dl were used for study¹⁷.

Table 1: Screening of phytochemicals in various extracts of C. asiaticum

Phytoconstituents/ Extracts	Petroleum ether	Chloroform	Ethyl acetate	Ethanolic	Aqueous
Alkaloids	-	+	-	+	+
Glycosides	-	+	+	+	+
Carbohydrates	-	-	-	-	-
Tannins and Phenolic	-	-	-	+	+
Flavonoids	-	+	+	+	+
Sterols	-	-	-	+	-
Proteins	-	-	-	-	+

Experimental Setup:

Animals were categorized into seven groups, each consisting of six rats. Standard pellet diet and water ad libitum was provided to the animals.

Group I: Normal healthy rats administered only vehicle (0.5% Tween 80)

Group II: Diabetic control (Alloxan 150 mg/kg)

Group III: Diabetic rats of this group were administered with glibenclamide (10 mg/kg) from 6^th day after first administration of alloxan

Group IV: Diabetic rats of this group were administered with C. asiaticum ethanolic extract (CAEE 200 mg/kg) from 6^th day after first administration of alloxan

Group V: Diabetic rats of this group were administered with C. asiaticum ethanolic extract (CAEE 400 mg/kg) from 6^th day after first administration of alloxan

Group VI: Diabetic rats of this group were administered with C. asiaticum aqueous extract (CAAE 200 mg/kg) from 6^th day after first administration of alloxan

Group VII:Diabetic rats of this group were administered with C. asiaticum aqueous extract (CAAE 400mg/kg) from 6^th day after first administration of alloxan

Oral glucose tolerance test¹⁸:

Prior to initiation of the experimental procedure, the rats were fed with a bolus of 2g/kg dose of glucose and the level of glucose in blood was estimated at 0, 30, 60 and 120 seconds after administration of glucose using glucometer.

Evaluation of antidiabetic activity:

The antidiabetic activity of the extracts was determined by measuring the blood glucose levels on 1^st, 10^th and 15^th day of administering the extract to the diabetic rats. The decline in glucose level was taken as the indicator for glucose ameliorating potential of the leaf extracts. Histopathological analysis of the pancreatic cells for degeneration and regeneration of islets of Langerhans was also performed by sacrificing the animal as per approved procedure and dissecting for isolation of the pancreas.

Statistical analysis:

The results of the animal experimentation are reported as mean ± standard error of the mean. Statistical difference between the means was determined by ANOVA followed by Dunnett’s test and p< 0.05 was accepted as significant.

RESULT AND DISCUSSION:

Cold maceration technique was employed to successively extract out the phyto-constituents of varying polarities from the leaves of C. asiaticum. The extraction yield followed the order aqueous > ethanolic > chloroform > petroleum ether > ethylacetate. The qualitative assessment of the phyto-constituents in the extracts of indicated the existence of flavonoids, sterols, glycosides, alkaloids, tannins and phenolics, proteins and saponins in various extracts (Table 1).

The ash values, extractives and loss on drying of the powdered material were calculated as per the procedures and the results of the same are reported in Table 2.

Table 2 Physicochemical values of the leaf extracts of C. asiaticum

Characteristic	Percentage (%w/v)
Total ash	25
Acid insoluble ash	9.0
Water soluble ash	18
Alcohol soluble extractives	23
Water soluble extractives	32
Loss on drying	14.62

Diabetes was induced in experimental animal using 150 mg/kg dose of alloxan administered intra-peritoneal over a period of 5 days. Ethanolic and aqueous extracts were used for the study of oral glucose tolerance as well as for evaluating the antidiabetic efficacy against alloxan-induced diabetes in rats. The presence of phenolics, tannins, flavonols and phytosterols has been widely responsible for various pharmacological actions of plant extracts and hence only ethanolic and aqueous extracts out of all the extracts of C. asiaticum were considered for pharmacological evaluation. The effect of C. asiaticum extracts on tolerating the bolus of glucose (OGTT) is presented in Table 3 and the changes in levels of blood glucose in normal, diabetic and extract treated diabetic animals is presented in Table 4.

Table 3 Effect of ethanol and aqueous extract of leaves of C. asiaticum on OGTT

Groups	Treatment / dose	Blood glucose (mg/dl)
Groups	Treatment / dose	0 h	0.5 h	1.0 h	2 h
I	Normal control	89 ± 1.3	124 ± 1.2 (↑40.0%)	115 ± 1.4 ( ↑30.0% )	91 ± 0.9 (↑3.33%)
II	Glibenclamide, 10 mg/kg	232 ± 1.9	293 ± 2.26 (↑26.06%)	289 ± 1.5 (↑ 24.35%)	285.4 ± 2.6 (↑22.64%)
III	Control, 0.5% Tween 80	177.2± 0.8	206.9 ± 1.6 (↑16.57%)	220 ± 1.02 (↑23.88%)	184.1 ± 0.8 (↑3.85%)
IV	CAEE 200 mg/kg	164.3±0.9	210.6 ± 1.09 (↑27.84% )a	237.9 ± 0.9 (↑44.25%)	194.1 ± 0.78(↑17.91%)
V	CAEE 400 mg/kg	181.3±0.6	210.6 ± 1.05 (↑15.98%)a	246.3 ± 0.76 (↑35.46%)	187.9 ± 1.5 (↓3.6%)a
VI	CAAE 200 mg/kg	207.1±1.4	239.5 ± 0.6 (↑15.49%)	260.6 ± 0.56 (↑25.10%)	190 ± 0.96 (↓8.18%)
VII	CAAE 400 mg/kg	193.5± 2.1	^s225.7 ± 0.76 (↑6.47%)a	234 ± 0.11 (↑20.71%)	178.1 ± 0.7 (↓12.77%)

Values are represented as mean ± SEM for (n=6); **p<0.01, *p<0.05 and ^nsp>0.05 compared to control

Table 4 Effect of ethanolic and aqueous extract of leaves of C. asiaticum on blood glucose in alloxan-induced diabetic rats

Groups	Level of Blood sugar in Group (15 days) mg/dL (mean ± SD)
Groups	Initial	Day 1	Day 5	Day 10	Day 15
Normal Control	68.76 ± 6.02	63.03±9.31	64.68±9.83	65.00 ±7.39	63.46 ±5.86
Glibenclamide,10 mg/kg	247.74±8.83	260.26±14.73	283.83±4.76	307.18±8.07	311.26 ±4.71
Control, 0.5% Tween 80	248.83±8.38	250.47 ± 5.55	237.21±8.40	202.36±5.82	190.01±5.78
CAEE 200 mg/kg	246.02±3.87	247.63 ± 7.83	219.22±5.39	187.8 ± 8.20	176.12 ±9.28
CAEE 400 mg/kg	247.68±8.83	254.06 ± 4.96	237.86±8.82	212.21±3.31	180.83 ±4.55
CAAE 200mg/kg	249.82±4.88	254.55 ± 5.55	231.43±6.28	190.75±4.87	152.83±10.22
CAAE 400mg/kg	246.36±3.48	249.15 ± 8.12	215.95±4.50	188.08±7.89	178.19 ±8.66

Values are represented as mean ± SD for (n=6); **p<0.01, and ^nsp>0.05 compared to control.

A previous study also reveals that the ethanolic extract of C. asiaticum has the potential to reduce the oxidative stress and blood glucose levels in alloxan induced diabetic mice⁴. Our study reinstates the findings and provides evidence that both ethanolic and aqueous extracts of C. asiaticum have the capability to regenerate the β cells and thereby enhance the insulin levels causing a decrease in serum glucose concentration.

CONCLUSION:

The present investigation provides proof that the ethanolic as well as aqueous cold extracts of the leaves of Crinum asiaticum possess the potential to ameliorate the elevated serum glucose levels that are caused due to the depletion of the β cells by alloxan. Furthermore, a comprehensive study to investigate the possible mechanism of action of the extracts as well as standardization of the extracts to develop into a potent natural medication has to be undertaken.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Shambhunath Institute of Pharmacy, Jhalwa, Uttar Pradesh, India for providing the necessary facilities for the present investigation.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 05.10.2020 Modified on 23.11.2020

Research J. Pharm. and Tech 2021; 14(12):6359-6363.

DOI: 10.52711/0974-360X.2021.01099