INTRODUCTION:

Diabetes is a chronic disorder linked with the metabolism of carbohydrate, protein and fat due to absolute or relative deficiency of insulin secretion with or without varying degree of insulin resistance¹. It also associated with impaired glucose metabolism leads to ketoacidosis, which if untreated, can lead to fatal ketoacidosis^2,3.

It is a major disease characterized by derangement in carbohydrate, fat and protein metabolism⁴. This disease is categorized as metabolic disease and characterized by hyperglycemia that is an important factor in the development and progression of microvascular progressions⁵. Streptozotocin (STZ) treatment results in diabetes due to the destruction of beta-cells of the pancreas that secrete insulin⁶.

Insulin therapy is the most common method used for the management of diabetes mellitus, but several drawbacks such as insulin resistance, anorexia nervosa, brain atrophy and fatty liver were reported⁷. Most of the hypoglycemic agents used in allopathic medicines are reported to have side-effects including hematological coma and disturbances in liver and kidney⁸. This has necessitated the exploration and screening of medicinal plants with acclaimed therapeutic efficacies in diabetes mellitus management⁹.

Azima tetracantha (Salvadoraceae) is a well known medicinal herb, termed ‘Mulsangu’ in Tamil and 'Kundali' in Sanskrit. Root, root bark and leaves of Azima tetracantha (lam) are used with food as a remedy for rheumatism, diuretic and as stimulant¹⁰. Traditionally Indian medical practitioners use Azima tetracantha (lam) in inflammatory conditions, cough, asthma, small pox and diarrhoea^11,12. The major phyto-constituents reported in Azima tetracantha (lam) are azimine, azecarpin, carpine, isorhamnitine-3-O-rutinoside, friedelin, lupeol, glutinol and β-sitosterol^13,14. Azima tetracantha (lam) is reported to have antifungal¹⁵ antitumour¹⁶, antidiabetic¹⁷, antidiarrhoeal¹⁸ and hepatoprotective¹⁹ activities.

Azima tetracantha (lam) is a low, spinouts, highly branched bush, woody below but with pale green, herbaceous, almost quadrangular young branches. The leaves are in opposite to sub-opposite, decussate pairs. They are shortly petiolate, about 2x4cm long, entire, elliptic, acute, sharply mucronate, rigid, pale green with an acute base. Usually, there are two laterally placed spines in the axil of a leaf. The spines which morphologically represent the first pair of leaves of the auxiliary shoot are about three cm long, more or less, triangular in cross section, very sharp and with an indurate apex. The plant is dioeciously. The flowers are borne in the axils of leaves. Generally, there is cymes of three flowers in the axil of a leaf which is the upper branches, especially of the male plants become greatly reduced or even completely suppressed.

Uses:

The plant is used in indigenous medicines for rheumatism, microbial infections, diahorrea, inflammatory conditions, reduce lipid and as hepato-protective.

MATERIALS AND METHODS:

Collection of plants:

The aerial part (leaves) of Azima tetracantha (lam) was collected from the Panayur area of Madurai, Tamilnadu as raw material, during the second week of February 2015 and a voucher specimen is stored in C.L. Baid Mehta College of Pharmacy (001/ATL/CLBP) and the plant material was authenticated by a renowned botanist. About 500 g of coarse powdered leaf in 2.5 L water is boiled, cooled and filtered. The filtrate is evaporated to dryness in desiccator and stored in refrigerator (Yield- 26.5% w/w). The aqueous extract of Azima tetracantha (lam) (AEAT) was subjected to preliminary phytochemical analysis²⁰

Various extraction methods for isolation of constituents:

The whole plant will be subjected to shade drying and extraction with petroleum ether (60-80^oC) chloroform, Ethyl acetate and 80% ethanol in soxhlet apparatus by simultaneous extraction each for 72 hours. Concentrate the solvents in vacuum. The crude solid obtained on evaporation are to be studied for preliminary qualitative phytochemical evaluation.

Phytochemical Screening:

The extract was subjected to phytochemical analysis to test the presence of carbohydrates, glycosides, alkaloids, flavonoids, tannins, sterols, and saponins in leaf extracts.

Preparation of animals:

Adult female albino rats weighing about 150-180 g were obtained from the animal house were used for the study. Rats were housed in polycarbonate cages in a room with a 12 hrs day-night cycle, at constant temperature of 22°C and humidity of 45-64%. During the experimental study rats were fed on pellets (Gulmohur Rat Feed, Lipton India, Bengaluru) with free access to tap water. The rats received humane care according to the criteria outlined in Principles of Laboratory Animal Care, 1985. The study was approved by Institutional Animal Ethics Committee (IAEC) and the experiments were conducted according to the ethical norms and IAEC guidelines.

Acute toxicity:

The toxicity study was carried out using thirty-five (35) male wistar rats weighing 180-200 g each. The animals were randomly distributed into one control group and six treated groups, containing five animals per group. They were maintained on animal cubes , provided with water ad libitum and were allowed to acclimatize to the laboratory conditions for seven days before the experiment after overnight fasting , the control group received normal saline and treated group received ethyl acetate extract of AT at the doses of 0.5, 1, 1.5,2, 2.5 and 3 g/kg body weight. The calculated doses were prepared with 0.5% carboxy methyl cellulose (CMC) in distilled water. These doses were given by intraperitoneal route. The animals were observed continuosly for the first 4h and then each hour for the next 24 h and 6 hourly intervals for the following 48 h after administering the extract, to observe any death or changes in general behaviour and other physiological activities. Acute toxicity and gross behavioural screening were studied.

Chronic toxicity^21,22:

Animals were divided into two groups were , one was control and other was test group . Dose of 100 mg/kg body weight /day of EA AT was administered in drinking water to each animal in the test group. This dose was selected on the basis of anti-diabetic effect of the EA AT in the mice. The treatment was continued for a period of three months during which changes in body weight was observed. Mice were analyzed for body and organ weight changes , haematological studies, and serum biochemical parameters.

Glucose tolerance:

Overnight fasted animals were divided to group o three (n=6). 1 mL of Normal saline given orally to Group I. Concentrations of 50, 450 mg / kg of Azima tetracantha ethyl acetate extract was administered to Groups II and III, respectively. Glucose (2 g/kg body weight.). Blood was administered and blood samples were taken at 0.5 hr, 1hr, 2hr and 4hr time from the vein in glucose administration. Blood Glucose was estimated and the Data was used as a hypothetical reference to determine the extract dose level. This data used in evaluation effects of Azima tetracantha extract on diabetic rats^23,24.

Treatment Protocol:

Animals were divided in to four groups; six in each group received the treatment schedule as tabulated below.

Treatment schedule:

Group Study:

I Normal control (saline).

II Streptozotocin treated control (150 mg/kg.ip)

III Streptozotocin (150 mg/kg.ip) + Azima tetracantha ethyl acetate extract (50mg/kg.ip)

IV Streptozotocin (150 mg/kg.ip) + Azima tetracantha ethyl acetate extract (150 mg/kg.ip)

V Streptozotocin (250 mg/kg.ip) + Azima tetracantha ethyl acetate extract (250 mg/kg.ip)

Anti diabetic effects:

Treatment was given as per the protocol. Blood samples were collected at 2, 4, 6 and 8 hours intervals from the fasted rat’s tail vein prior to administration of the extract. Glucose levels were measured from the separated Serum. According to the protocol the non-fasted animals daily treated with ethyl acetate extract of Azima tetracantha for 18 days. Blood samples were collected on 6th day, 9th day, 12th, 15th and 18th day after Streptozotocin and glucose levels in blood serum were measured.

Liver and kidney functions:

ALP (Alkaline Phosphatase), creatinine, GPT (glutamate pyruvate transaminase) and BUN (Blood urea nitrogen) were measured. Protein content was determined by the method of . Total WBC count was determined by using a heamocytometer.

Antioxidant status:

GSH (reduced glutathione), GP (glutathione peroxidase), SOD (superoxide dismutase), catalase, and MDA (malondialdehyde) levels were determined in liver homogenates by spectrophotometrically using assay kits available commercially. Total nitrites present in aortic tissues were colorimetric ally estimated by using Griess reagent. Concentration of nitrite in the sample was calculated using sodium nitrite standard and normalized to the aorta protein content.

Statistical Analysis:

Statistical evaluation for all the grouped data performed by ANOVA. Values were expressed as mean ± SEM (Standard Error of Mean) for six animals in each group. Unpaired student t-test is used for statistical comparison between the four different groups. Changes were considered to be statistically significant if the P-value was < 0.05. +p < 0.05, ++p < 0.01, +++ p<0.001 and *p<0.0001 was considered statistically significant²⁵.

RESULTS:

Effects of Azima tetracantha ethyl acetate extract in the single dose study on blood glucose levels was estimated after Streptozotocin administration on the 3rd day. There is no reduction in glucose level due to Azima tetracantha ethyl acetate extract (50 mg/kg). At dose levels of 150 and 250 mg/kg, glucose levels were decreased by 24.8% and 29.9% at the 6th hour, respectively. Anti diabetic effect of the extract was slightly decreased at the 8th hour, but remained statistically significant (Table-1).

In parallel experiments, Azima tetracantha ethyl acetate extract was administered to diabetic rats for 18 days as per the protocol. The glucose level reduction up to 26% on 9th day of Azima tetracantha ethyl acetate (50 mg/kg) treatment started to lower serum glucose on the 9th day. An overall reduction of 26% was observed on the 18th day by 50mg/kg administration (p<0.01). 52.5% reduction of glucose level in the serum absorbed at 18th day administration at dose level of 150 mg/kg (p<0.0001). 250mg/kg extract administration is more effective from 6th day onwards with a reduction rate of 18.9% (p<0.01) and maximum reduction of serum glucose level by 61.9% on the 18th day. Between the 12th and 18th days, anti diabetic effect of Azima tetracantha ethyl acetate was in a concentration dependent manner(Table-2).

The effects of Azima tetracantha ethyl acetate extract on hepatic and renal function in Streptozotocin-diabetic rats. As seen, treatment with 50 mg/kg did affect neither the significantly high levels of ALP, GPT, BUN and creatinine, nor the overall oxidative status. Conversely, rats treated with higher doses of the extract (150 and 250 mg/kg) showed significant improvements in hepatic and renal function (Table-3). Additionally, these two dose levels recovered the weight loss and low white blood cell count observed in Streptozotocin-diabetic rats while decreasing liver glycogen (Table-4). Azima tetracantha ethyl acetate extract (150 and 250 mg/kg) also showed a protective effect on liver oxidative status (Table-5).

Antioxidants namely GSH, GPx, SOD and catalase were increased by Azima tetracantha ethyl acetate extract administration. When compared to Streptozotocin-diabetic rats, MDA formation, as an indirect measure of lipid peroxidation, was found to be significantly low in high dose Azima tetracantha ethyl acetate extract treated rats.

The ethyl acetate extract was screened for Streptozotocin-induced anti diabetic activity. The ethyl acetate extract of plant showed significant anti diabetic activity at both doses, that is, 150 and 250 mg/kg of body weight. This is further evidenced by percentage reduction in blood glucose levels after 18th day after administering the extract at both of the doses. The ethyl acetate extract significantly increased the body weight of diabetic animal at higher doses.

DISCUSSION:

In the present study, Azima tetracantha Lam was selected for anti diabetic studies owing to its traditional uses. Therefore, the study was undertaken to justify its claimed uses. Wistar rats were selected as experimental animals for the anti diabetic activity. The extract was screened for Streptozotocin-induced anti diabetic activity. The ethyl acetate extract of plant showed significant anti diabetic activity at both doses, that is, 150 and 250 mg/kg of body weight. This is further evidenced by percentage reduction in blood glucose levels after 18th day after administering the extract at both of the doses. The ethyl acetate extract significantly increased the body weight of diabetic animal at higher doses. During this prolonged study, various physical parameters were also observed such as body weight, food intake, water intake, and weight of internal organs. Generally, body weights are reduced in diabetic animals, but in this study, the decrease in body weights was diminished by the extract treatment; thus this effect may be useful for the diabetic animals. The phytochemical study showed the presence of saponin glycosides, steroids, and flavanoid compounds in the extracts, which might be a reason for the good activity of extract. However, this is a preliminary work, and more work is needed to determine the active ingredients in the extract

which may help in improving management of the anti diabetic agents. The study reveals that the ethyl acetate extract of Azima tetracantha Lam could be added in list of herbal preparation, beneficial in diabetes mellitus. Azima tetracantha Lam can be considered as an important addition to the therapeutic treatment of diabetes. The present investigation has also opened avenue for further research especially with reference to the development of potent formulation for diabetes

mellitus from Azima tetracantha Lam.

Table-1: Effects of Azima tetracantha on serum glucose levels in Streptozotocin –

induced diabetic rats

Group

Serum glucose level (mg/dL)

0 hour

2 hour

4 hour

6 hour

8 hour

70.5

± 5.6

67.2

± 6.9

64.0

± 7.9

67.4

± 7.1

69.2

± 6.9

267.9

± 10.5_a

274.1

± 9.7_a

277.0

± 0.9_a

273.1

± 8.0_a

267.4

± 7.6_a

III

261.4

± 9.6

264.4

± 8.6

258.2

± 8.5

255.2

± 9.9

252.5

± 8.7

258.2

± 8.5

253.0

± 8.1

237.4

± 7.6

204.9

± 9.2_b

214.2

± 9.0_c

259.9

± 10.9

241.9

± 9.9

230.5

± 7.9

191.2

± 8.4_b

198.3

± 8.5_b

After administration of Streptozotocin the serum glucose levels were obtained from fasted rats. Data are expressed as mean ± S.E; (n=6); ap<0.0001 (compared to normal group with the corresponding hours). bp<0.01 and cp<0.05 (compared to control group with the corresponding hours)

Table -2: Effects of Azima tetracantha (Daily treatment) on serum glucose levels in

Group

Serum glucose (mg/dL)

3 days

6 days

9 days

12 days

15 days

18 days

74.9

± 7.3

72.8

± 8.7

71.3

± 10.6

74.1

± 8.8

70.8

± 6.8

75.0

± 8.3

272.4

± 13.0^#

279.4

± 11.6 ^#

271.5

± 11.6^#

273.7

± 11.0^#

268.6

± 11.0^#

263.0

± 9.5^#

III

269.9

± 10.9

249.6

±11.0

230.5

± 9.4⁺

218.5

± 9.1⁺

208.7

± 9.9⁺

199.7

± 9.1⁺

275.3

± 9.7

231.4

± 10.5

211.1

± 12.3⁺⁺

174.3

± 10.0⁺⁺⁺,a

151.0

± 10.7⁺⁺⁺,b

131.4

± 11.4_*,c

271.5

± 10.6

220.7

± 9.3⁺⁺

166.4

± 11⁺⁺⁺

141.4

± 8.8_*,d

130.8

± 9.6_*,e

104.6

± 12.3_*,f

Values of serum glucose levels were obtained from Streptozotocin induced diabetic rats in the absence and in the presence of 18 days of Azima tetracantha ethyl acetate extract treatment (from the 3rd to the 18th day) and expressed as mean ± S.E; n=6; #p<0.0001 (compared to normal group with corresponding day); +p<0.05, ++p<0.01, +++p<0.001 and *p<0.0001 (compared to control group with corresponding day). ap<0.05, bp<0.01 and cp<0.001 (compared to Group III with corresponding day). d,e,f p<0.05 (compared to Group IV with corresponding day)

Table-3: Effects of Azima tetracantha ethyl acetate extract on liver and kidney functions

Group	Liver		Kidney
Group	ALP (KA/dL)	GPT (U/mg protein)	BUN (mg/dL)	Creatinine (mg/dL)
I	34.9 ± 4.0	160.6 ± 11.3	10.4 ± 1.8	1.9 ± 0.2
II	51.5 ± 7.9_a	336.5 ± 23.1b	20. ± 3.4c	3.3 ± 0.6.c
III	43.8 ± 6.0	286.8 ± 21.0	17.6 ± 12.3	2.9 ± 0.4
IV	36.5 ± 3.9_e	245.4 ± 13.5_e	14.8 ± 1.9_f	2.7 ± 0.8_e
V	32.3 ± 13.3_f	207.3 ± 10.3_f	12.7 ± 1.8_g	2.4 ± 0.6_g

ALP: alkaline phosphatase; GPT: glutamate pyruvate transaminase; BUN: blood urea nitrogen. Liver and kidney markers were measured on the 18th day after Streptozotocin administration. Data are expressed as mean ± S.E; n=6; ap<0.01, bp<0.001 and cp< 0.0001; compared to normal group. ep<0.05, fp<0.01 and gp<0.001 (compared to control group)

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Received on 27.09.2018 Modified on 20.10.2018

Research J. Pharm. and Tech 2019; 12(2):660-664.

DOI: 10.5958/0974-360X.2019.00117.3

Group	Body Weight		Total leukocyte count (mm3)	Liver glycogen (μg/g tissue)
Group	Initial	Final	Total leukocyte count (mm3)	Liver glycogen (μg/g tissue)
I	240.5 ± 6.6	258.2 ± 7.9	13233.4 ± 455.5	75.7 ± 3.4
II	244.1 ± 8.4	204.1 ± 20.7_a	8566.0 ± 388.0_c	56.9 ± 3.6_d
III	248.4 ± 8.9	244.4 ± 18.6_b	9451.4 ± 510.0	66.6 ± 2.9
IV	242.2 ± 6.9	248.0 ± 11.1	11102.4 ± 656.9_e	73.3 ± 3.9_e
V	246 ± 10.9	261.9 ± 7.9	13001.3 ± 701.9_f	76.4 ± 4.3_e

Parameter	Group I	Group II	Group III	Group IV	Group V
GSH (nmol/mg protein)	9.6±1.6	5.4±1.4_c	5.7±1.5	7.1±1.6⁺	7.5±1.6⁺⁺⁺
GSSG (nmol/mg protein)	1.5±0.2	2.1±0.3c	1.9±0.3	1.8±0.6⁺	1.5±0.4⁺⁺⁺
GPx (U/mg protein)	1.4±.0.16	1.3±0.5_a	1.3±0.3	1.2±0.1⁺	1.1±0.1⁺
MDA (nmol/mg tissue)	355.4±11.4	443.4±3.1_b	421.5±14.3	391.4±10.4⁺	356.8±13.8⁺⁺
SOD (U/mg protein)	7.5±0.6	4.5±0.5_c	4.8±0.5	5.6±0.8⁺⁺	6.8±0.7⁺⁺⁺
Catalase (U/mg protein)	154.6±7.9	99.5±8.2_b	120.4±9.5	127.4±8.7⁺	143.9±11.8⁺⁺