INTRODUCTION:

Medicinal plants consist of components of therapeutic values and have been used as remedies for human diseases since long. Recently, due to the pathogens resistance against the available antibiotics and the recognition of traditional medicine as an alternative form of health care has reopened the research domain for the biological activities of medicinal plants¹. Medicinal plants being as an important natural resource and potentially safe drugs can play an important role in assuaging human health by contributing herbal medicines. In the rural and remote areas of India, more than 70 percent of population depends on folk and traditional system of medicines obtained from plants. The high cost of allopathic medicine and their potential side effects, encouraged the people to use the traditional medicine².

Diabetes mellitus is a principal cause of morbidity and mortality in human populations³. It is a syndrome characterized by hyperglycemia, polydipsia and polyuria and causes complications to the eyes, kidneys, and nerves. It is also associated with an increased incidence of cardiovascular disease⁴. It is the most common endocrine disorder, affecting approximately 16 million individuals in the United States and as many as 200 million worldwide. Perhaps more importantly, individuals with diabetes are disproportionate users of the health care system. In 1992 health care expenditures per individual with diabetes were more than three times greater than per capita expenditure for those without diabetes.

Ficus is a pan-tropical genus of about 850 species of woody trees, shrubs, vines, epiphytes, and hemiepiphyte in the family Moraceae. Collectively known as fig trees or figs, they are shrubs or trees, sometimes scandent, sap milky leaves alternate, rarely opposite, entire, rarely serrate or lobbed, stipules varous. The use of Ficus species as ethnomedicine in Nepal is quite noteworthy⁵. F. benghalensis (Bar) is most important, used to heal 22 ailments. Ficus sarmentosa bark powder is taken to cure boils and secrete more milk during delivery. Root extract is used in malaria⁶. A preliminary study in our laboratory showed decreases in blood glucose levels and food intake in STZ-diabetic rats given aqueous or ethanolic extracts of leaves of B. variegata, F. palmata and F. carica intraperitoneally⁷. However, no reports are available about the constituent(s) of these plants which is responsible for the anti-diabetic activity and the mechanism (s) of their anti-diabetic action. Hence the aims of this study is to investigate the effects of various extracts of the leaves of F. carica on blood glucose and lipid levels in STZ- diabetic rats and to investigate the anti-diabetic effects of these extracts in rat model of type 2 diabetes.

MATERIALS AND METHODS:

Following solvents, chemicals and reagents of analytical grade or best possible grade supplied by approved companies were used.

Solvents/Reagents Used:

Table 1: List of chemicals

S. No.	Name	Supplier
1.	Chloroform	Ranbaxy Labs, New Delhi.
2.	Methanol	Ranbaxy Labs, New Delhi.
3.	Ethanol	Ranbaxy Labs, New Delhi.
4.	Petroleum Ether	Ranbaxy Labs, New Delhi.
5.	Carbon tetrachloride	Ranbaxy Labs, New Delhi.
6.	Tween 80	Ranbaxy Labs, New Delhi.
7.	Starch	Ranbaxy Labs, New Delhi.
8.	Sodium CMC	Ranbaxy Labs, New Delhi.
9.	Sucrose	Ranbaxy Labs, New Delhi.
10.	Sodium lauryl sulphate	Ranbaxy Labs, New Delhi.
11.	Microcrystalline cellulose	Ranbaxy Labs, New Delhi.
12.	Dexamethasone	Ranbaxy Labs, New Delhi.

Equipment and Instruments Used:

Glucometer:

Wockhardt Ltd. Mumbai

Experimental Animal selection:

The albino Wistar rats (male and female) weighing more than 130gm were procured from animal house of Bharat Technology, Howrah and maintained at standard house conditions, fed with commercial feed and water ad libitum. All the housing conditions and animal handling were in accordance with the CPCSEA-OECD guidelines. Before performing the experiment, the ethical clearance was obtained from the Institutional Animal Ethical Committee, Bharat Technology, Howrah.

Collection and Authentication:

In the present study, the leaves of Ficus carica were collected from local area of District Chamoli, Uttarakhand. The plants were authenticated from Botanical Survey of India, and voucher specimens were preserved at Herbarium of Bharat Technology, Howrah.

Extraction of Plants:

The shade dried leaves of Ficus carica were reduced to fine powder (# 40 size mesh) and around 500g of powders were subjected to successive hot continuous extraction (soxhlet) with petroleum ether, ethyl acetate, methanol and water. After the effective extraction, the solvent was distilled of, the extracts were then concentrated on water bath and the extract obtained with solvent was weighed. Its percentage will be calculated in terms of air-dried weight of plant material. The colour and consistency of the extracts will be noted. For aqueous extract maceration was used.

Experimental Induction of Diabetes:

The animals were selected, weighed and marked for individual identification. After fasting for 18-h, the rats were intraperitoneally injected with a single dose of 60 mg/kg STZ, freshly dissolved in citrate buffer (0.01 M, pH 4.5). Diabetes in the rats was identified by polydipsia, polyuria and by measuring non-fasting serum glucose concentration 48-h after injection of STZ. Rats with a serum glucose level above 300mg/dL were selected for experiments. Normal control rats were injected with saline only. After one hour of STZ administration the animals were given feed ad libitum. A 5% dextrose solution was given bottle for a day to overcome the early hypoglycaemic phase. The diabetic rats (glucose level > 300mg/dl) were separated and divided into different groups for experimental study, each group comprised of six animals^8,9,10.

Acute Oral Toxicity- Acute Toxic Class Method:

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co-operation and Development (OECD), received draft guidelines 423, received from Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. The method enables a judgment with respect to classifying the test substances to one of the series of toxicity classes defined by fixed LD₅₀cut off values.

Healthy young albino rats of either sex weighing between 150 to 220g (8 to 12 weeks old) were used for acute toxicity study to determine LD50 of various extracts. Totally there were ten groups, each group consisting of three animals. The animals were randomly selected, marked to permit individual identification, and kept in their cages for 5 days prior to dosing to allow for acclimatization to the laboratory conditions. The temperature in the experimental room was around 25°C. Lightening was artificial, the sequence being 12 hrs. dark and rest 12 hrs. light. The conventional laboratory diet was feed, with an unlimited supply of drinking water¹¹.

The extract was prepared as a suspension by triturating with water and 1% Tween 80. The test substances were administered in a single dose by intra gastric tube. Prior to dosing, animals were kept for 12 hours of fasting. Then animals were weighed and test substance was administered. After the dose was administered food was withheld for a further 3-4 hours. In each step three animals were used in each group. Study was begun at 300mg/kg body weight and continued up to 5000mg/kg body weight. 1/10^th of this lethal dose was taken as effective dose (therapeutic dose) for subsequent pharmacological screening.

Animals were observed initially after dosing at least once during the first 30 minutes, then periodically during the first 24 hrs. In all cases mortality was observed with in the first 24 hrs. Additional observations were noted like changes in skin and fur, eyes, and mucous membranes and behaviour pattern. Observation was also made for tremors and convulsions^11,12.

Experimental model:

Albino Wistar rats weighing more than 160 gm were used for the experiments. Prior to dietary manipulation, all rats were fed standard pellet rodent diet and water ad libitum and maintained on a 12-hour light/dark (06:00/18:00) cycle. After acclimatization, the rats were divided into different groups of six each. All groups of animals received treatment for 9 days. Food was given to the animals twice daily in the morning and evening. The daily body weight has been measured. The Normal control group animals received food and vehicle only while different extract groups received the drug before the food by intragastric tube at 10.00am. Glibenclamide was given in the form of aqueous suspension to the Diabetic standard control group animals, once in a day by intragastric tube. The volume of the suspension was 1ml/100gm of rat.

Table 2: Experimental protocol

Group	Diabetic animals	Non diabetic animals	OGTT animals
1	Normal control (NC) (Vehicle only)	Normal control (NC) (Vehicle only)	Normal control (NC) + Glucose (2g/kg)
2	Diabetic control (DC)
3	Aqueous extract 500mg (AEFS)	Aqueous extract 500mg (AEFS)	Aqueous extract 500mg (AEFS) + Glucose (2g/kg)
4	Methanolic extract 500mg (MEFS)	Methanolic extract 500mg (MEFS)	Methanolic extract 500mg (MEFS) + Glucose (2g/kg)
5	Ethyl acetate extract 500mg (EAFS)	Ethyl acetate extract 500mg (EAFS)	Ethyl acetate extract 500mg (EAFS) + Glucose (2g/kg)
6	Petroluem ether extract 500mg (PEFS)	Petroluem ether extract 500mg (PEFS)	Petroluem ether extract 500mg (PEFS) + Glucose (2g/kg)
7	Glibenclamide 600µg/kg (GLB)	Glibenclamide 600µg/kg (GLB)	Glibenclamide 600µg/kg (GLB)

AEFS: Aqueous extract of F. carica

MEFS: Methanolic extract of F. carica

EAFS: Ethyl acetate extract of F. carica

PEFS: Petroleum ether extract of F. carica

Preparation of Glibenclamide drug solution:

Glibenclamide in its pure form was obtained from Sun pharmaceuticals Ltd., Selaqui, Dehradun, India. 1ml aqueous solution was given to diabetic standard control group (as a 600µg/kg body weight) daily using an intragastric tube for 9 days.

Collection of blood sample:

The blood sample was collected from the rat-tail vein. During the period of study and at the end of the treatment period, 2 drops of blood were taken by rat vein after 16 hours fasting and spread on the marked end of Pulsatom blood gluco-strip and the strip is inserted in the gluco-meter.

After few seconds the gluco-meter displayed the blood glucose level which was noted down.

Body Weight Measurement:

Body weight has been measured totally four times during the course of study period i.e., on and before STZ induction (initial values), 1^st day, 4^th day and 9^th day of the treatment period, using a digital weighing scale obtained from ORION Engineering weighs.

Oral glucose tolerance test:

Fasted rats were divided into four groups of six rats each. Group I served as normal control and received distilled water with Tween 80. Groups II to XIII received different extracts at a dose of 500mg/kg as a fine Tween 80 suspension. Group XIV received the standard drug Glibenclamide as an aqueous suspension at a dose of 600µg/kg body weight.

After 30 min of extract administration, the rats of all groups were orally treated with 2g/kg of glucose. Blood samples were collected from the rat tail vein just prior to glucose administration and at 30, 60 and 90 min after glucose loading. Blood glucose levels were measured immediately by using Gluco-meter^13,14.

Statistical Analysis:

Results were expressed as the mean ± SEM. Statistical analysis was carried out using one-way analysis of variance followed by Neuman-keul’s test for comparison between groups. P<0.05 was considered as significant.

RESULT:

In the present work, three plants were selected on the basis of literature survey and traditional uses from the local area of Chamoli (Garhwal) to screen anti-diabetic potential. It was observed that all the plants were extensively used in the treatment of variety of diseases.

Toxicity studies:

Results of acute toxicity studies of all the extracts of individual plants are given in Table 10 and effective dose is mentioned in Table 11. Toxicity studies were conducted on albino Wistar rats. Acute toxicity study was carried out according to OECD guidelines. Starting dose was selected to be 2000 mg/kg body weight up to 5000 mg/kg body weight as specified for natural products. At 5000 mg/kg weight, no mortality was observed which is considered as the end point. So, 1/10^th of this dose was taken as experimental dose for subsequent antidiabetic studies. For the aqueous, methanolic, ethyl acetate and petroleum ether extracts of FS, the effective dose was taken on the basis of previously published reports, i.e., 500 mg/kg body weight.

Table 3: LD₅₀ Cut off mg/kg body weight of various extracts

S. No.	Name of Extract	LD₅₀ Cut off mg/kg body weight	Vehicle + Suspending agent
1	Aqueous extract (AEFS)	5000 mg	Tween 80
2	Methanolic extract (MEFS)	5000 mg	Tween 80
3	Ethyl acetate extract (EAFS)	5000 mg	Tween 80
4	Petroluem ether extract (PEFS)	5000 mg	Tween 80

Antidiabetic activity:

Four different extracts of four different plants were screened for antidiabetic activity in STZ induced diabetic rats. Results of antidiabetic activity of FS are mentioned in Table 4.

Table 4: Antidiabetic activity of leaves extracts of Ficus carica.

S. No.	Blood Glucose level mg/100ml
	Normal Control		Diabetic Control		Glibenclamide
	Basal value	9th Day	Basal value	9th Day	Basal value	9th Day
1	73	80	333	347	316	138
2	79	78	345	336	339	144
3	76	79	338	341	342	130
4	76	81	326	335	323	147
5	69	78	340	342	333	135
6	75	79	335	338	341	192
Mean	74.67±1.382	79.17±0.477	336.2±2.651+++	339.8±1.81+++	332.3±4.349	139.2± 2.52***
±SD	3.386	1.169	6.494	4.446	10.65	6.178

Continue Table 4

S. No.	Blood Glucose level mg/100ml
	*Ficus sarmentosa* Extracts
	Petroleum ether		Ethyl acetate		Methanol		Aqueous
	Basal value	9th Day	Basal value	9th Day	Basal value	9th Day	Basal value	9th Day
1	314	386	327	153	316	140	332	151
2	324	370	320	152	327	252	347	178
3	317	310	316	189	340	162	313	147
4	316	363	319	216	326	185	352	163
5	319	359	327	280	329	140	335	150
6	345	340	324	162	320	182	327	163
Mean	334.3± 9.265	320.0± 7.58	347.8± 10.37	220.7± 27.2***	339.2± 15.42	208.5± 34.9***	322.5± 4.43	201.8± 22.1***
±SD	22.7	18.59	25.4	66.82	37.77	85.54	10.86	54.3

Initially blood glucose level was estimated 72 hrs after STZ administration and 9^th day of the treatment with the extracts for all animals. Blood glucose levels were expressed as mg/100ml (mg%) and were given in mean ± standard error.

As expected, administration of alloxan led to elevation of fasting blood glucose levels (p<0.001), which was maintained over a period of study in diabetic control group and 9 days of daily treatment 500mg/kg body weight with aqueous, methanolic, ethyl acetate and petroleum ether extracts of FC was given. Ethyl acetate and petroleum ether extracts FC, aqueous led to normalization of blood glucose levels. The effect seemed to reach maximum on first day itself and gradually restored nearer to normal level in subsequent days.

Ethyl acetate and petroleum ether extract of FC treated group were able to reduce blood glucose level significantly as compared to diabetic control. The aqueous and methanolic extracts of FC treated groups reduce the blood glucose level significantly when compared to diabetic control.

Aqueous, methanolic and ethyl acetate extracts of FC treated groups were able to reduce blood glucose level significantly as compared to diabetic control. The aqueous extract of FC has shown more significant reduction in blood glucose level compared with other extracts of the same plant. The petroleum ether extract of FC showed least significant reduction in blood glucose level compared with other extracts of same plant. Oral treatment with standard hypoglycemic agent Glibenclamide (GLB) 600µg/kg body weight was also able to reduce the elevated BGL towards the normal.

Body Weight Measurement:

Normal vehicle control animals were found to be gained in their body weight but diabetic control rats showed significant reduction in the body weight, which is reserved by extract treated groups during 9 days study. The treatment with petroleum ether, ethyl acetate, methanol and water extracts of FS (500mg/kg body weight) showed increase in the body weight compared with the Diabetic control (132.2±1.81, 126.5±1.08 and 110.3±2.37) in 1^st, 4^th, and 9^th day of study. In FS, the aqueous extract showed increase in body weight while methanolic extract showed minimum increase in body weight. Glibenclamide treated group (150.7±1.43) group also prevented this reduction in body weight.

Oral Glucose Tolerance Test (OGTT):

The effect AEFS, MEFS, EAFS and PEFS on glucose tolerance is shown in Table in table below. By 30 min after starting the glucose tolerance test, the blood glucose concentration increased rapidly from its initial value as was evident from normal control, but the extracts treated groups (with 500mg/kg body weight of EAFS and PEFS,) prevented significantly glucose-induced hyperglacemian at 30 min and 90 min as compared to that of normal control at 30 min and 90 min (183.2±3.68 and 135.7±2.917). Glibenclamide treated group (600µg/kg) also prevented significantly glucose induced hyperglycemia at 30 min and 90 min (170±0.57 and 105.0±0.57) as compared to normal control (184.7±0.66 and 104.8±0.94). Maximum glucose tolerance was observed in aqueous extract (106.5±2.1) and minimum glucose tolerance was observed in petroleum ether extract of (121.7±10.06**) FS in 90 minutes compared with the normal control.

Table 5: OGTT test

S. No.	*Blood Glucose Level mg/100ml (Mean±SEM) (Ficus sarmentosa)*
	Normal Control Glucose (2g/kg) (NC)			Glibenclamide Glucose (2g/kg) (NC)			Petroleum ether
	FBGL	30 min	90 min	FBGL	30 min	90 min	FBGL	30 min	90 min
1	86	197	129	86	187	110	72	171	113
2	84	186	139	89	168	112	73	171	114
3	89	174	126	75	170	101	72	171	113
4	82	177	145	82	174	98	74	171	113
5	79	176	135	69	179	105	72	171	113
6	80	189	140	74	183	108	72	171	113
Mean	83.33± 1.54	183.2± 3.68	135.7± 2.917	79.17± 3.15	176.8± 3.04	105.7± 2.20**	72.67± 2.87	171.0± 12.9	113.2± 6.45**
± SD	3.78	9.02	7.15	7.73	7.47	5.39	7.03	31.75	15.82

Continue table 5

S. No.	*Blood Glucose Level mg/100ml (Mean±SEM) (Ficus sarmentosa)*
	Ehtyl acetate			Methanol			Aqueous
	FBGL	30 min	90 min	FBGL	30 min	90 min	FBGL	30 min	90 min
1	79	168	104	75	179	106	79	189	121
2	78	169	104	68	179	106	82	210	108
3	77	186	103	73	178	105	85	172	103
4	78	168	104	84	179	106	69	159	110
5	78	170	104	89	180	107	81	147	109
6	79	168	104	92	179	107	86	191	100
Mean	78.33± 2.70	168.0± 6.05	104.3± 2.09**	80.17± 3.919.57	179.3± 4.95	106.5± 5.54**	80.33± 2.49	178.0± 9.43	108.5± 2.95**
± SD	6.62	14.83	5.12	9.57	12.13	13.58	6.12	23.12	7.23

DISCUSSION:

In the present study three medicinal plants were screened for the antidiabetic activities in rats. The selected plant parts were collected from the local area of Chamoli, Garhwal i.e., leaves of Ficus carica was authentified from Botanical Survey of India, Dehrahun and voucher specimens were preserved at Bharat Technology, Howrah .

All the parts were subjected to size reduction to get coarse powder and then passed through sieve no. 40 to get uniform powder. Then the uniform powder was subjected to standardization with different parameters as per Pharmacopoeias/literature. After physicochemical characterization, all the parts were successively extracted with petroleum ether, ethyl acetate, methanol, and water for acute toxicity study and chemical studies, antidiabetic activity and antihypertensive activity.

Phytochemical investigation revealed the presence of different chemical constituents like steroids, saponins, alkaloids, proteins, sugars, flavonoids, tannins, phenols, amino acids and steroidal glycosides in different plant extracts. Aqueous, methanolic and ethyl acetate extracts of FC had decreased blood glucose level. The effect seems to reach maximum on first day itself and gradually restored to normal level in subsequent days^15,
16.

There was a significant elevation in BGL in STZ induced diabetis control (p<0.001) rats when compared with normal control. Oral treatments with 5000mg/kg body weight of aqueous, methanolic and ethyl acetate extracts of FC reduced blood glucose level significantly as compared to diabetic control.

Oral glucose tolerance test was performed on non-diabetic rats after challenging with glucose. Results indicated that ethyl acetate and methanolic extract of FC showed significant tolerance.

The measurement of body weight was made in order to observe the effect of various extracts on the change in body weight. It was observed that ethyl acetate and methanolic extract of FC showed minimum increase in body weight in diabetic rats.

CONCLUSION:

Diabetes mellitus is one of the most common global health concerns, with a rapidly increasing incidence. A variety of medicinal plants, particularly those belonging to the genus ficus (Moreaceae) and their active compounds have been used to treat diabetes and related chronic disorders since ancient time [17]. The present study demonstrated that the ethyl acetate, methanolic and aqueous extract of Ficus carica leaves reduced blood glucose level and body weight significantly. This may justify the use of ficus species as ethanomedical medicine for treatment of diabetes mellitus.

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Received on 11.06.2020 Modified on 28.10.2020

Research J. Pharm. and Tech. 2021; 14(8):4151-4156.

DOI: 10.52711/0974-360X.2021.00718