INTRODUCTION:

Diabetes mellitus is an endocrine disorder that is characterized by hyperglycemia.¹ This disease is major degenerative ailment in the world today and has affected at least 15 million people.² The management of diabetes mellitus is considered a global problem and successful treatment is yet to be discovered.³ Throughout the world a great number of plants have been suggested as a rich as yet unexplored scientific source of potentially useful anti-diabetic drugs. However, the ultimate objective of their use is that they should interact directly with our body chemistry with out side effects.^4-5 Synthetic hypoglycemic agents those are capable of reducing blood sugar level possessed most worrying side effects. Therefore, finding other anti-diabetes agents, especially those made from natural sources is desired.⁶

Caesalpinia pulcherrima belonging to Fabaceae family is considered as one of the most promising plant in Indian System of Medicine for treating various diseases. The plant is an ornamental plant widely distributed throughout India. The plant shrub having varying medicinal properties, which are widely used for treatment of inflammation, ulcer, fever, tumors etc. Traditional Caesalpinia pulcherrima has been in clinical use in India since ancient times.

MATERIALS AND METHODS:

Caesalpinia pulcherrima is a species of flowering plant in the pea family, Fabaceae. Caesalpinia pulcherrima popularly known as Guleture (Hindi), Peacock flower (English) and Ratnagandhi (Sanskrit) found in India. It is a shrub or small tree up to 5m in height abundantly cultivated. Widely distributed in the tropics and subtropics region, Native to Americas, West Indies and is also distributed in all over India but its exact origin is unknown due to widespread cultivation. The flowers are borne in racemes up to 20cm long, each flower with five yellow, orange, or red petals. Leaves are bipinnate, 20-40 cm long bearing 3-10 pairs of pinnate with 6-10 pairs of leaflets 15-25mm long broad. Traditionally Caesalpinia pulchirrima is used as purgative, tonic, antipyretic, emmenagogue, whereas roots have folkforic use in convulsion, intermittent fever, lungs and skin diseases Flavonoids are polyphenolic compounds, widely distributed in the plant kingdom. They are reported to exhibit various pharmacological activities such as CNS, cardiotonic, lipid lowering, anti-oxidant, hepatoprotective and hypoglycemic activities. The wood is reported to contain glycosides containing β-amyrin, glucose and the free amino acid: alanin, glycine, valin; free sugar, lactose, galactose and glucose also present.

Selection of plant and authentication:

Dried aerial parts of plant Caesalpinia pulcherrima were collected from the campus of NIET, Greater Noida, during the long stretch of September 2018 and it was authenticated by Dr. Anjula Pandey (Taxonomist), National department of plant and hereditary assets (NBPGR), Pusa grounds, New Delhi. A voucher (specimen no: NHCP/NBPGR/2019-22) is protected in herbarium segment of ordered branch of NBPGR, New Delhi. The shade dried stem aerial part of Caesalpinia pulcherrima plant were mixed in the ratio of (1:3) and extraction was carried out by double cold maceration process with hydroalcololic extract solvent for 7 days with occasionally shaking every 4 hours and placing them in dark condition so as to minimize the light entrapment and maintaining the proper temperature 35⁰C. After 3 days filtration was carried out and marc was re-macerated with the solvent for another 3 days. The filtration was concentrated in rotatory evaporator and water bath by maintaining the temperature not exceeding 35⁰C with continuous stirring. The extract was concentrated to a liquid form and placed in a container. The dried extract was found to be 43.34% w/w with regard to airdried drug.

Preparation of Dose:

The dose of (10mg/kg) of standard metformin is prepared by using sterilized water, Caesalpinia pulcherrima extract was dissolved in 10% DMSO to prepare dose at a concentration of 200mg/kg and 300mg/kg body weight.

Induction of Diabetes:

The five groups, containing six animals in each group were taken. Diabetes was induced experimentally in rats by a single intraperitoneal injection of a freshly prepared solution of streptozotocin (STZ) at a dose of 75mg/kg body weight (bw) in normal saline. After 48 hr, blood was collected with all aseptic precautions from the tail vein of the rats under mild ether streptozotocin (STZ blood was collected with a anesthesia and blood glucose levels were determined using glucometer (infopia Co., LTD.). The animals were considered to be diabetic if the blood glucose values were more than 180mg/dl, and those animals alone were used for the study. Diabetes was developed and stabilized in STZ treated rats over a period of 7 days. Control rats were given normal saline. The hypoglycemic activity of Caesalpinia methanol extract was assessed by feeding the test extract (200/300 mg/kg, p.o, bw/day) to extract treated diabetic animals. The animals were followed up to 5 h to check the time required for the test extract to produce peak hypoglycemic activity. The animals were divided into five groups of six animals each.

GROUP I: Normal saline control.

GROUP II: Diabetic control received STZ only.

GROUP III: Diabetic induced rat treated with standard hypoglycemic agent, metformin (10mg/kg bw/day), P.O

GROUP IV: Diabetic induced rats treated with Caesalpinia pulcherrima aerial plant extract (200 mg/kg), P.O

GROUP V: Diabetic induced rats treated with Caesalpinia pulcherrima aerial plant extract (300 mg/kg), P.O

The rats were followed for 5 hr, with the blood sugar levels being estimated at the end of 1hr, 3 hr, and 5 hr. Test drug treatment was continued for 12 weeks, at the end of which the rats were sacrificed with marked response liver were isolated and carried out histopathologically.

Statistical Analysis:

All results were expressed as mean± SEM. The data were analyzed using analysis of variance (ANOVA), and the group means were compared by Dunnett's test. Values were considered statistically significant with P<0.05. GraphPad Instat was used for the analysis of data.

RESULTS:

Effect of CPAE extract (200 and 300 mg/kg) on blood glucose level in STZ- induced diabetic rats model:

To study the antidiabetic potential of Caesalpinia pulcherrima, The extract at the dose of 200 and 300 mg/kg in Wistar rats for 45 days. In animals, after the administration of STZ, diabetes was induced due to pancreatic β-cell islet cytotxicity. In this study, treatment with Caesalpinia pulcherrima (200 and 300mg/kg) significantly (P < 0.001) reduced the elevated blood glucose levels from first to fourth week when compared with diabetic control rats. The Caesalpinia pulcherrima at a dose of 300mg/kg significantly (P< 0.001) produced more reduction of blood glucose than its lower dose of 200mg/kg. There are several studies present to support the different mechanisms behind antidiabetic plants to produce their blood glucose reducing effect, such as elevated insulin release and secretion, damaged pancreatic islet β-cells regeneration, insulin sensitivity enhancement and carbohydrate metabolizing enzymes inhibition. The Caesalpinia pulcherrima may exert its hypoglycemic activity possibly by above mentioned mechanisms and the antidiabetic activity of Caesalpinia pulcherrima was comparable to that of Metformin.

Table 3: Hepatoprotective activity of aerial parts of CPAE on STZ induced Diabetic rat

Groups	Drug	Dose mg/kg	AST (IU/L)	ALT (IU/L)	Total Protein	ALP (IU/L)	Total Bilirubin (mg/dL)
1	Saline	0.2ml	27.81±1.98	28.18±0.84	3.04±0.59	81.16±0.59	0.77±0.005
2	STZ	0.75	6.29±0.81	35.94±0.93	5.44±0.21	88.85±0.27	0.81±0.01
3	Metformin	10	16.11±0.63**	22.35±0.53** *	6.99±0.04***	76.04±0.86***	0.70±0.04***
4	CPAE	200	19.34±0.71*	25.32±0.53*	6.47±0.32*	75.50±0.86*	079.001*
5	CPAE	300	17.20±0.54**	23.37±0.74**	7.04± 0.35**	75.88± 0.02	0.05± 0.07

Values were expressed in MEAN ± SEM (n=6), **p<0.1 and ***p<0.01 is highly significant when compared with control group (one way ANOVA followed by Dennett’s t test): it represented as Caesalpinia pulcherrima hydroalcoholic extract, AST: Aspartate aminotransferase, ALT: Alanineaminotransferase, ALP: Alkalinephosphate.

Hepatoprotective activity:

The rise in the AST and ALT in diabetes induced hepatotoxicity and the increased level of AST and ALT in group V (post treatment with extract at the dose of 200mg/kg was essentially lessened. Metformin was discovered noteworthy diminishment in the stage of AST and ALT when contrasted with the control.

Calculated values of hepatoprotective activity of CPAE on STZ induced Diabetic rat. (Table-3).

Histopathological findings:

(A) STZ induced control (B) Standard (Metformin)

(C) CPAE extract: 200 (D) CPAE extract: 300

Figure 3: Effect of CPEA on histopathology of liver in diabetes rat

Histopathology:

Photomicrograph of rat liver section (staining with haematoxylin)

(A) Marked increase in aggregation of hepatocytes and necroinflammatory changes after STZ administration. (B) Liver section after administration standard drug (Metformin) at the dose of 10mg/kg showing reduced hepatocellular necrosis, ballooning degeneration and inflammatory, (C) Liver sections after adding extract (200mg/kg) showing moderate improvement of inflammatory cells and cell necrosis. (D) Liver sections after adding extract (300mg/kg) showing reduced necrosis area and increased number of hepatocytes.

CONCLUSION:

Diabetes Mellitus is one of the most common disorders which affect millions of people worldwide. Current therapies have increased the quality of life and life span of the diabetic patients but these medicines are only helpful in managing this disease and not treating it. Type- 2 diabetes mellitus increases the blood glucose level by decreasing the mass and function of pancreatic β-cells. Over the year, the medicinal property of natural plants has been the main focus of the research and development of plant based therapeutic strategies is a cost effective approach which provides medicines with least side effects. Similarly, previous studies have demonstrated tha Caesalpinia pulcherrima can attenuate insulin resistance as well as metabolic disturbances in experimental model of diabetes and hepatoprotective. Caesalpinia pulcherrima: is a multiorgan-multipurpose plant which means that the different part of the plant have shown different pharmacological activities. Our study for the first time showed anti-diabetic and hepatoprotective property of extract CAPE at a dose of 200 and 300mg/kg per oral in STZ induced diabetic rat model.

In the current study, streptozotocin treatment induced diabetic rats which were displayed by an increased level of plasma blood glucose and alteration in several serum haematological markers. CAPE at both 200mg/kg and 300mg/kg showed a dose- dependent reduction of blood glucose level of diabetic rats thereby exhibiting its antidiabetic property. This activity was confirmed by CAPE mediated reversal of the modified effect of various haematological markers. CPAE again dose- dependently showed increase in Hb and total protein level as well as a decrease in HbA1c and AST level. This observed hypoglycemic activity of the aerial parts of the plant can be due to the combined effect of the compound present in the extract. The possible mechanism of the CAPE mediated hypoglycemic activity can be due to the potentiation of pancreatic β-cells insulin secretion and/or due to enhanced transport of blood glucose to the peripheral tissue or may be by some other mechanism such as stimulation of glucose uptake by peripheral tissue, inhibition of endogenous production of glucose or activation of gluconeogenesis in liver and muscle.

Some Hepatotoxic drugs such as over dose of iron, chloroform known to cause checked height in serum level chemicals, for example, AST, ALT, ALP, total protein and total billirubin showing noteworthy hepatocellular damage. The estimation of compound in serum is valuable quantitative markers of the concentrate and sort of hepatocellular harm.

In diabetes rat levels of AST (36.29±0.81) IU/L, ALT (35.94±0.93) IU/L, ALP (88.85±0.27), total billirubin (0.81±0.01) mg/dL in serum was significantly increased when compared with control. The CPAE on 200mg/kg and 300mg/kg dose treatments decreased the level of AST [(19.34±0.71) and 17.20±0.54) IU/L], ALT [(25.32±0.53) and (23.37±0.74) IU/L], ALP[ (78.50±0.86) and (75.88±0.02) IU/L], and total billirubin [(0.79±0.01) and (0.75±0.07) mg/dL] when compared STZ control. In this study, CAPE reduced SGOT and SGPT that is plant can be considered as a agent for the treatment of liver toxicity.

Metformin (10mg/kg, body weight) treated animals also showed significant decreased in AST (16.11±0.63) IU/L, ALT (22.35±0.62) IU/L, ALP (76.04±0.85) IU/L, total billirubin (0.70±0.04) mg/dL when compared with diabetes treated rat.

The present examination expanded levels of serum transaminase in investigational rat, is seen can be credited to the harmed structural intregity of the liver because these are cytoplasmic in nature and are free into the course after cell hurt. Decrease in the absolute serum protein was assessed in rat treated with STZ might be connected with the lessening in the quantity of hepatocytes, which thus may impact in the hepatic ability to deliver protein and therefore diminish liver burden. Organization of methanol concentrate of Caesalpinia pulcherrima indicated critical hepatoprotective action, which was contrasted and the standard medication (Metformin). Hepatocellular putrefaction or on the other hand membrane harm lead to extremely abnormal amounts of serum AST, ALT discharged from liver to flow, abnormal state of billirubin respectively.

Therefore concluded that the hepatoprotective activity of Caesalpinia pulcherrima rationalize the conventional state of the plant for the hepatoprotective activity. It was observed from the study that aerial parts of Caesalpinia pulcherrima extract. Caesalpinia pulcherrima has revealed the capability to regulate the normal function of the liver Caesalpinia pulcherrima showed the potent hepatoprotective properties in STZ induced diabetic rat model.

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Received on 11.02.2020 Modified on 05.04.2020

Research J. Pharm. and Tech. 2021; 14(7):3716-3720.

DOI: 10.52711/0974-360X.2021.00643

Groups	Blood Glucose Level mg/dl
Groups	Day 1	Day 14	Day 28	Day 45
Normal Control	57.11 ± 1.5	67.10 ± 1.5	62.44 ± 1.5	70.46 ± 2.8
Diabetic Control	329.65 ± 6.5***	341.60 ± 6.19***	357.87 ± 6.3***	341.33 ± 7.4***
CPAE 200mg/kg	319.61 ± 6.5	273.64 ± 8.6*	171.59 ± 8.8**	115.28 ± 7.3***
CPAE 300mg/kg	313.77 ± 5.9	181.35 ± 9.5**	101.72 ± 5.5***	90.86 ± 6.3***
Metformin 10mg/kg	324.22 ± 5.2	193.75 ± 11.6**	95.56 ± 5.5***	88.44 ± 5.9***

Group	Hb (g/dl)	HbA1c (%)	Total protein (g/dl)	SGPT
Normal Control	15.09 ± 1.4	6.76 ± 0.73	6.7 ± 0.75	33.56 ± 0.76
Diabetic Control	10.15 ± 1.1***	13.66 ± 1.08***	5.11 ± 0.58***	139.12 ± 7.5***
CPAP200	13.98 ± 1.4**	5.77 ± 0.64***	6.47 ± 0.75**	102.04 ± 5.4*
CPAP 300	14.84 ± 1.4***	4.93 ± 0.55***	6.28 ± 0.72**	70.64 ± 2.8***
Metformin	15.32 ± 1.4***	5.92 ± 0.57***	6.83 ± 0.77***	62.32 ± 1.3***