In Vivo Validation of Folkloric Use of Costus pictus D. Don as Antidiabetic Plant in Assam, Using Streptozotocin Induced Wister Rat Model

Abstract

Costus pictus D. Don belonging to the family Costaceae is one of the most commonly used plant among traditional healers in the upper Assam region in India, specifically used in the treatment of diabetes. Aerial parts of the plant are said to have potent anti diabetic property. The present study was aimed to evaluate the traditionally claimed antidiabetic activity of aerial parts of C. pictus in animal models. Healthy male Wister rats (120 ± 30 gm) were used in the study and diabetes was induced by Streptozotocin (STZ) i.p. prepared by dissolving in citrate buffer (pH 4.5), along with nicotinamide (120 mg/kg/b wt). Diabetic rats were treated for 14 days with daily doses of methanolic extract of C. pictus (MECP) in three different scheduled amounts (50, 100 and 200 mg/kg/b wt; p.o.). Control rats were treated with 0.3% CMC (Carboxy Methyl Cellulose) suspension (10 ml/kg/b wt; p.o.). Blood glucose level and plasma lipid profile was examined on 1st, 7th, 14th and 21st day 1 h after treatment. On the last day, 1 h after the treatment, animals were sacrificed followed by isolation of pancreas and liver for histopathological examination. The results were compared with that of the standard group treated with Glibenclamide (10 mg/kg/day; p.o). Comparison of the experimental data of different cohorts demonstrated the potential antidiabetic activity of C. pictus however the highest dose of 200 mg/kg/b wt; p.o. of MECP significantly (P < 0.05) reversed the STZ induced diabetic parameters (increased blood glucose level, altered plasma profile and histoarchitecture of the pancreatic and hepatic cells) that is comparable with that of the standard. The observed results suggest anti diabetic efficacy of C. pictus thereby uphelding the folkloric usage.

Introduction

The North Eastern part of India is a rich depository of folklore. This part of India is a hot spot of cultural and biological diversity. The North East region of India comprises eight states that harbor more than 180 major tribal communities of the total 427 tribal communities found in India [1]. The state of Assam, popularly known as the land of the red river and blue hills is the gateway to the North East India. Geographically the state extends from 22°19′ to 28°16′ North Latitude and 89°42′ to 96°30′ East Longitude between the foot hills of the Eastern Himalayas and the Patkai and Naga Hill Ranges. In Assam around forty languages are spoken by different communities belonging to major language families like Austro-Asiatics, Sino-Tibetan and Indo European. The ethnic diversity among the different communities is also observed in the treatment of different ailments by utilization of the abundant natural resources [2, 3].

Costus pictus D. Don belonging to the family Costaceae (Fig. 1) is one of the most commonly used plant cutting across ethnic background among traditional healers in the upper Assam region which is specifically used in the treatment of diabetes [4]. The aerial parts of the plant is used for compounding traditional formulation and prescribed accordingly to the patients. Costus being the largest genus of the family Costaceae with over 100 species, mostly indigenous to the neo-tropical region of South America, Mexico and West Indies and about 20 species are also found in Asia and Africa [5–8].

Fig. 1.

C. pictus D. Don collected from Naojan, Golaghat, Assam

Botanically, C. pictus is an erect plant, up to 3 m high; root stock is tuberous; stem is sub-woody at the base. Leaves are elliptical and are spirally arranged around the stem. Leaf surface is firm and leathery. The primary bracts are borne on the inflorescence in spiral phyllotaxy. Flowers are yellow in colour with pink radiating stripes originating from the base. Flowering time in Indian condition is August to October [9, 10].

Plants belonging to the family Costaceae are mostly used for febrile conditions, coughs, skin conditions, retention of the placenta, post-partum bleeding, threatening abortion, insufficient uterine contractility and snake bites [11].

The present study was therefore aimed to validate the traditionally claimed antidiabetic activity of aerial parts of C. pictus D. Don in animal models.

Materials and Methods

Collection and Authentication of Plant Material

The aerial parts of C. pictus D. Don were collected from Naojan, Golaghat during the month of September–October. The plant was identified and authenticated taxonomically vide Letter No. BSI/ERC/Tech./2015–16 at Botanical Survey of India, Shillong.

Preparation of Plant Materials

The aerial parts were air-dried under shed for 2–6 week time. It was grinded to make fine powder in a blender and then packed in air tight containers for extraction and evaluation.

Pharmacological Evaluation

Selection of Animals

Healthy Wister rats weighing between 120 and 150 gm were used for the study. The in vivo study was approved by IAEC (Institutional Animal Ethical Committee) of Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam-786004 (Regd. No. 1576/GO/a/11/CPCSEA Dated: 17/02/2012) vide Approval No: IAEC/DU/36 Dated: 03/12/2012.

Maintenance and Acclimatization of Animals

The animal house was well ventilated and animals were kept under conditions of 24 ± 20 °C and RH 44–56% and 12 h light: 12 h dark cycles each day for 1 week before and during the experiments. Animals described as fasted were deprived of food for 16 h but allowed free access to water.

Preparation of C. pictus D. Don Extract (MECP)

The crude drug was extracted with methanol using hot continuous process and concentrated to dryness in rotary vacuum evaporator.

Acute Toxicity Studies

Acute toxicity study was performed for methanolic extract of C. pictus (MECP) as per protocol drawn under OECD guidelines 425 in female Wister rats. Limit test at 2000 mg/kg was conducted as per section 23 of OECD guideline 425 [12]. Animals were fasted overnight and maintained with water ad libitum. Dosing of one animal was done at the test dose of 2000 mg/kg. The animal was observed continuously to detect changes in the autonomic or behavioural responses and also for tremors, convulsion, salivation, diarrhoea, lethargy sleep and coma and then monitored for any mortality for the following 14 days. A group of animals treated with vehicle alone served as control.

The animal did not show any mortality including overt toxicity signs of tremors, convulsion, salivation, diarrhoea, lethargy sleep and coma during the entire observation period.

The doses of 50, 100 and 200 mg/kg body weight respectively were selected for the study.

Antidiabetic Activity Study

Induction of Diabetes

Diabetes was induced in the rats by single injection (i.p.) of Streptozotocin (STZ), prepared by dissolving in citrate buffer (pH 4.5), at a dose of 60 mg/kg body weight along with nicotinamide at a dose of 120 mg/kg body weight after overnight fasting. Nicotinamide was simultaneously administered along with STZ so as to prevent STZ from completely destroying the β cells of the pancreas thereby sustaining a Type II diabetes model. Animals with FBS ≥ 200 mg/dl were then considered for the study.

Animal Model

Healthy Wister rats (120 ± 30 gm) were divided randomly into six groups containing six animals each as mentioned in Table 1. The duration for the experimentation procedure was 3 weeks i.e. 21 days. Group I served as the control, and received the vehicle (0.3% CMC suspension, 10 ml/kg/b wt; p.o) for 21 days. Group II served as the negative control group, and received Streptozotocin (STZ) (i.p.) 60 mg/kg body weight and Nicotinamide (NIC) 120 mg/kg body weight. Diabetic rats were treated with daily doses of MECP, at a dose of 50, 100 and 200 mg/kg b wt/p.o. respectively. Group III served as test group with lower dose and received MECP, at a dose of 50 mg/kg b wt/p.o. daily. Group IV served as test group with higher dose which received MECP at a daily dose of 100 mg/kg b wt/day/p.o. and Group V served as test group with highest dose, and received MECP at a daily dose of 200 mg/kg b wt/day/p.o. for 21 days. Lastly, Group VI served as the standard control group and received Glibenclamide at a dose of 10 mg/kg/day; p.o for 21 days.

Table 1.

Animal distribution chart for in vivo antidiabetic activity study

Group	No. of animals	Vehicle	Drug/dose/route	Name of group
I	6	0.3% CMC suspension	10 ml/kg/b wt; p.o	Vehicle control (+ve control)
II	6		STZ 60 mg/kg + NIC 120 mg/kg b wt; i.p.	STZ induced (−ve control)
III	6		MECP (50 mg/kg p.o.)	Test dose 1
IV	6		MECP (100 mg/kg p.o.)	Test dose 2
V	6		MECP (200 mg/kg p.o.)	Test dose 3
VI	6		Glibenclamide (10 mg/kg p.o.)	Standard control

Estimation of Serum Biochemical Parameter

Blood was collected from marginal ear vain during the study period and allowed to stand for 20 min, and centrifuged for 15–20 min at 2000 rpm to separate the serum and the latter was used for serum cholesterol and triglyceride estimation.

Total Cholesterol Estimation

Total Cholesterol was estimated using CHOD/POD method [13]. The absorbance of the standard and test sample against blank was measured at a wavelength of 505 nm. Total Cholesterol content was then measured using the following formula,

Cholesterol (mg/dl) = (Abs. T/Abs. S) × 200 mg/dl; where T denotes test, S denotes standard and Abs denotes absorbance. 200 mg/dl is the concentration of standard used.

Serum Triglyceride Estimation

Serum Triglyceride was estimated using GPO or GOD–POD method [14]. The absorbance of the standard and test sample against blank was measured at a wavelength of 500 nm. Triglycerides content was then measured using the following formula,

Triglycerides (mg/dl) = (Abs. T/Abs. S) × 200 mg/dl; where T denotes test, S denotes standard and Abs denotes absorbance. 200 mg/dl is the concentration of standard used.

Estimation of Blood Glucose

Blood glucose level was examined on 1st, 7th, 14th and 21st day of the experimental procedure, 1 h after treatment of standard and test drug. The FBS were recorded using Accu-Chek ^® Active blood glucose monitoring system.

Histopathology

Following method was followed to prepare the liver slides for observation:

Stage 1: Tissue Processing

Tissues were at first cleared by washing with acetone. Tissue samples were kept in acetone for 0.5 h. The whole process is repeated for three times.

In this step the tissues were subjected to dehydration. Samples were kept in benzene for about 0.5 h. It is also repeated for three times.

Stage 2: Embedding

Paraffin was melted in oven for about 2 h. Tissue samples were embedded in the molten paraffin and made into blocks. Then the blocks were allowed to cool. After that the blocks were placed in microtome and ribbon strips were sectioned with a thickness of few millimeters. The slides were then incubated overnight at a temperature of 37 °C.

Stage 3: Staining

Slides were melted in spirit lamp and were washed with xylene for 3–5 min, repeated twice. Then washed again with absolute, 90% and 70% alcohol for 3–5 min repeating each washing twice. After that the slides are washed under running water for about 20 min.

In this step the slides were stained with haematoxylin, the latter was kept for about 3–5 min for proper tissue penetration. The slide appeared bluish in colour. After that the excess stain was washed off under running water.

The slides were then treated with acid–alcohol (1% HCl in absolute alcohol). The slide now appears violet. Then the slides were washed under running water for about 2–3 min. Then these are treated with eosin for 30 s.

Stage 4: Dehydration and Fixing

Dehydration was done with the help of alcohol treatment followed by xylene. Xylene treatment was done for about 3–5 min and the process was repeated thrice. After that these were mounted in DPX and covered with cover slip. The slides were then observed under microscope.

On the last day, 1 h after the treatment animals were sacrificed followed by isolation of pancreas and liver for histopathological examination as per above mentioned method and observed under microscope (Leica Photomicroscope DM1000) [15–17].

Results and Discussion

Serum Biochemical Parameter Estimation

The effects of MECP on investigated serum biochemical parameter viz. total cholesterol and serum triglycerides were investigated in STZ induced diabetic rats using Glibenclamide as standard. The results are summarized in Table 2.

Table 2.

Effects of C. pictus D. Don on total cholesterol and serum triglycerideslevel on experimental animals

Groups	Total cholesterol (mg/dl)*	Serum triglycerides (mg/dl)*
G-I Vehicle control	109.37 ± 12.41	115.25 ± 6.05
G-II STZ induced	185.25 ± 17.36	218.50 ± 18.53
G-III Test 1 (50 mg/kg)	142.75 ± 4.85	167.92 ± 10.60
G-IV Test 2 (100 mg/kg)	131.25 ± 7.45	153.50 ± 14.56
G-V Test 3 (200 mg/kg)	124.04 ± 10.23	131.43 ± 8.21
G-VI Standard drug	117.50 ± 18.27	122.17 ± 9.81

*Values are mean ± SEM (n = 6). Values are statistically significant at (P < 0.05)

Blood Glucose Level Estimation

The comparative effects of different doses of MECP with respect to the standard drug in streptozotocin induced diabetic rats on fasting blood sugar (FBS) was examined on 1st, 7th, 14th and 21st day 1 h after treatment using Accu-Chek ^® Active blood glucose monitoring system. The results are summarized in Table 3.

Table 3.

Effects of different doses of MECP on blood glucose levels in the treatment period

Animal Group	Blood glucose concentration in mg/dl*
	1st day	7th day	14th day	21st day
G-I Vehicle control	112.62 ± 3.12	105.44 ± 4.04	108.12 ± 3.03	116.49 ± 3.86
G-II STZ induced	298.21 ± 12.81	306.88 ± 13.05	316.48 ± 13.11	323.64 ± 14.05#
G-III Test 1 (50 mg/kg)	312.61 ± 26.53	311.12 ± 13.53	308.50 ± 24.66	314.21 ± 18.65
G-IV Test 2 (100 mg/kg	289.09 ± 11.63	280. 05 ± 15.15	282.63 ± 8.91	278.15 ± 9.66
G-V Test 3 (200 mg/kg	305.01 ± 13.81	278.89 ± 18.60	206.64 ± 12.22	123.89 ± 9.60#
G-VI Standard drug	294.41 ± 6.53	264.15 ± 7.12	142.80 ± 4.05	109.81 ± 6.61#

*Values are mean ± SEM (n = 6)

^#Values are statistically significant at (P < 0.05)

Histopathological Study

Liver and pancreas were isolated from each of the sacrificed animals and processed using rapid method as described in the histopathology section above. The histopathological slides were physically observed for any changes effected by the different treatment of test as well as the standard drug on the chemically induced diabetic rat models. The results of histopathological studies on liver and pancreas are depicted in Figs. 2 and 3 respectively.

Fig. 2.

Comparative changes in histo-architecture of liver with different doses of test (MECP) and standard drug as observed under Leica Photomicroscope DM1000 at 40 × magnification

Fig. 3.

Comparative changes in histo-architecture of pancreas with different doses of test (MECP) and standard drug as observed under Leica Photomicroscope DM1000 at 40 × magnification

Discussion

MECP could successfully alter the biomarker levels; particularly the 200 mg/kg/b wt dose closely resembles standard group in successfully controlling the elevated cholesterol and triglyceride levels. MECP 200 mg/kg treated animals significantly (P < 0.05) reversed STZ induced diabetic parameters (increased blood glucose level, altered plasma profile and histoarchitecture of the pancreatic and hepatic cells) that is comparable with the standard drug. It can be concluded that MECP has significant role in reversing altered lipid profile and blood glucose level in diabetic rat model.

Conclusion

Costus pictus D. Don was evaluated on the basis of consolidated data regarding the usage of the plant in treating diabetes among healers practicing traditional medicine particularly in the upper Assam region. Studies on animal models pertaining to evaluation of antidiabetic and anti hyperlipidemic profile of the plant was initiate to substantiate the folkloric use of the plant in the treatment of diabetes. In vivo study on streptozotocin induced diabetic rats was undertaken at three dose levels viz. 50, 100 and 200 mg/kg b wt p.o. and the results were compared with that of glibenclamide (10 mg/kg p.o.), which was used as a standard. The histopathological as well as blood and serum parameter data firmly established the anti diabetic potential of C. pictus D. Don thereby upholding the traditional belief regarding the utility of the said plant in treating diabetes. The results were found to be significantly comparable, particularly at the highest test dose of 200 mg/kg b wt p.o. with that of the standard drug used for the study. The histopathology of STZ treated rats showed microvesicular steatosis and occasional macrovesicular steatosis in prevenular area of hepatic lobules along with sinusoidal dilatation and mild congestion. The portal triads reveal mild increase in inflammatory infiltrate of mononuclear cells and neutrophils. The overall hepatic architecture was maintained according to findings of the experiments. Therefore, it can be summarized that the aerial parts of C. pictus D. Don is of considerable pharmacological importance and hence justified its use in diabetes treatment in the traditional treatment system.

The promising results warrant further study towards isolation and identification of bio-active principles from MECP as a future prospective.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving animals were in accordance with the ethical standards promulgated by the Institutional Animal Care Committee, CPCSEA, India. The institutional registration number is 1576/GO/a/11/CPCSE Dated: 17/2/2012. The present study is approved vide Approval No. IACE/DU/36 Dated 03/12/2012.