Quisqualis indica Linn was reported for treatment of various diseases like flatulence26, coughs, diarrhoea28, body pains, worm infection, toothache35, cardiovascular system26. Immuno modulatory activity of Quisqualis indica Linn was due to the presence of flavonoids, vitamin C, or the carotenoids27. A number of pharmacological studies such as immunomodulatory41, larvicidal46, and nematicidal47, antibacterial, antioxidant43, antipyretic, anthelmintic44 and antirhumatic properties31 have been reported on Quisqualis indica Linn. The present work deals with the preliminary screening of the various phytochemicals, their extraction, and qualitative estimation of phytoconstituents, isolation, structure elucidation and analytical method development for the same.
The morphological studies of Quisqualis indica Linn revealed that the leaf is dark green color with characteristic odour and slight bitter taste. The shape of Quisqualis indica Linn leaves is elliptical-acuminate with entire margin, cordate base, and length varying from 7-12 cm. Dorsal side is glabrous and ventral surface is hairy and Quisqualis indica Linn flowers have raceme type arrangement, texture is very smooth and silky. The flowers are fragrant and tubular and their color varies from white to pink to red, average size of flower is about 30-35 cm long. Microscopic studies of leaves showed the presence of covering and glandular trichomes. Midrib is having hypodermis which is made up of collenchymas. Lamina showed the presence of chlolenchyma next to epidermis. Midrib region showed xylem towards upper epid ermis. Protoxylem found to move towards upper epidermis and meta xylem towards lower epidermal cells, Phloem moves towards lower epidermis. Microscopic studies of flower showed glandular and covering unicellular trichomes on epidermis, oil glands, pericyclic fibers, xylem and parenchymatous tissue in middle of ovary.
Get your grade
or your money back
using our Essay Writing Service!
Powder study revealed the presence of covering trichomes, annular xylem vessel, epidermal cell, and anomocytic stomata. The stomatal index was 15.12 - 20.32, stomatal number was 0.28 - 0.32, vein islet number was 2-5, vein termination was 3-6, phloem fibers (6.76 - 78.21 length, 1.06 - 1.45 width), Ca-oxalate was (1.6 - 3.2 length, 1.2 - 1.6 width), trichomes (10.62 - 45.54 length, 1.56 - 2.56 width) and starch gains 1.56-8.0 diameter.
The moisture content, total ash, acid insoluble ash, water-soluble ash and sulfated ash values of leaf were observed to be 12 %, 7.3 %, 1.5 %, 3.5 % and 5.67 % w/w respectively where as physico-chemical constant for flower were found to be 10 %, 5.2 %, 1.4 %, 3.3 % and 3.45% w/w respectively. Water-soluble extractive, alcohol soluble extractive values of leaves and flowers were found to be 19.4 %, 16.6 %, and 13.2 %, 12.1 % w/w respectively.
For the purpose of isolation various extracts using different solvents were prepared and screened for the identification of various phytoconstituents. Successive solvent extraction was carried out with the two solvents namely petroleum ether and methanol. The extractive values were found to be more in the methanol extract. The petroleum ether extracts of leaves and flowers showed the presence of sterols, terpenoids, fixed oils and fats. Methanol extracts showed the presence of flavonoids and phenols. The pet-ether fraction gave positive Libermann-Burchard test indicating the presence of phytosterols. The pet-ether extract was further subjected to fractionation with solvent ether to separate unsaponifiable matter in order to get phytosterols enriched fraction. The unsaponifiable matter fraction of leaves were loaded separately on silica column and eluted with Benzene - Methanol in gradient elution mode. The collected fractions of leaves were grouped together into five fractions on the basis of their TLC profiles. Compound La was crystallized out from fraction PEL1 and 2. It was further purified by washing with methanol. Fraction PEL-4 on repeated washing with methanol it afforded compound Lb. The unsaponifiable matter fraction of flowers were loaded separately on silica column and eluted with Chloroform - Methanol in gradient elution mode. The collected fractions of the flowers were grouped together into five fractions on the basis of their TLC profiles which showed homogeneity in different mobile phases. Compound Fa was crystallized out from fraction PEF A, B and it was further purified by washing with methanol. Fraction PEF-C on repeated washing with methanol afforded compound Fb.
MLQ and MFQ were subjected to partition with ethyl acetate and water. The ethyl acetate fraction of leaves (3.1%) was subjected to column chromatography on silica gel (60-120 mesh, 1.5 kg) column. Gradient elution was carried out using ethyl acetate and increasing the polarity with methanol in stepwise increments. to yield two fractions. According to the similarity of the TLC profile the fraction MLQ-3, 4 and 5 were concentrated. The compound Lc and Ld were separated by preparative TLC method. The ethyl acetate fraction of flowers (2.3%) was subjected to column chromatography on silica gel (60-120 mesh, 1.5 kg) column. Gradient elution was carried out using chloroform, ethyl acetate and increasing the polarity with methanol in stepwise increments to yield two broad fractions according to the similarity of the TLC profile. The fraction EF-1, 2, 4 and 5 were concentrated. The compound Fc and Fd were separated by preparative TLC method. The fraction was purified by preparative TLC, after concentration, it formed yellow precipitate. The precipitation showed positive result for shinoda test and homogeneity with different mobile phases. The one phenolic compound was obtained from ethyl acetate insoluble fractions of methanolic extracts of leaves and flowers by column chromatography which shows positive result with ferric chloride. In the present study isolation of five phytoconstituents each from leaves and flowers of Quisqualis indica Linn. The compound isolated from leaves and flowers were combined on the basis of chemical test, Rf value and color shown by the spot on TLC plate after spraying.
Always on Time
Marked to Standard
The flowers and Leaves each contain same five compounds (i.e. A, B, C, D and E) which were isolated and confirmed by using chromatographic techniques. These compounds were subjected for structural elucidation by using different spectroscopic methods.
Compound A (Î²- sitosterol): Î» max from UV spectrum indicated the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups like hydroxyl (-OH) stretch, C-H stretch of alkenes and C=C stretch for cycloalkenes. 1H NMR and 13C NMR showed aliphatic protons and hydroxyl proton and presence of 29 carbons in structure. The molecular weight of compound (m/e 414) corresponding to the molecular formula C29H50O which was confirmed by literature. A comparative study of spectroscopy data with the literature revealed that compound A was steroidal terpenoids, Î²- sitosterol.
Compound B (Lupeol): Î» max from UV spectrum indicates the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups of hydroxyl (-OH) stretch, C-H stretch of alkenes and C=C stretch for cycloalkenes. 1H NMR and 13C NMR showed aliphatic protons and hydroxyl proton and presence of 30 carbons in structure. The molecular weight of compound (m/e 426) corresponding to the molecular formula C30H52O which was confirmed by literature. A comparative study of spectroscopy data with the literature revealed that compound B was pentacyclic terpenoid as Lupeol.
Compound C (Quercetin): Î» max from UV spectrum indicates the presence of conjugation and two chromophores which have specific character of flavonoids. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=O stretch for lactone and aromatic benzonoid ring. 1H NMR and 13C NMR showed aromatic protons and hydroxyl proton and presence of 15 carbons in structure. The molecular weight of compound (m/e 302.24) corresponding to the molecular formula C15H10O7 which was confirmed by given literature. A comparative study of spectroscopy data with the literature revealed that compound C was phenyl propanoid flavanol as Quercetin.
Compound D (Rutin): Î» max from UV spectrum indicates the presence of conjugation and two chromophores which have specific character of flavonoids. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and C=O stretch for lactone and aromatic benzonoid ring. 1H NMR and 13C NMR showed aromatic protons and hydroxyl proton and presence of 27carbons in structure. The molecular weight of compound (m/e 609) corresponding to the molecular formula C27H30O16 which was confirmed by literature. A comparative study of spectroscopy data with the literature revealed that compound D was phenyl propanoid flavanol as Rutin.
Compound E (Gallic acid): Î» max from UV spectrum indicates the absence of conjugation and chromophore. FT-IR spectra resulted in presence of functional groups hydroxyl (-OH) stretch, C-H stretch of alkenes and aliphatic carbon, C=O stretch and C-O stretch. 1H NMR and 13C NMR showed aromatic protons and hydroxyl proton and presence of seven carbons in structure. The molecular weight of compound (m/e 168) corresponding to the molecular formula C7H6O5 which was confirmed by given literature. A comparative study of spectroscopy data with the literature revealed that compound E was phenolic compound as Gallic acid.
Phenolic or polyphenol compounds constitute the main class of natural antioxidants present in plants, foods and beverages and have received considerable attention because of their physiological functions, including antioxidant, antimutagenic, antitumor activities101, 102. Direct free radical scavenging action of phenol in serviceable food is responsible for antioxidant activity103. Results showed the total phenolic content was found to be 0.0067 and 0.0056 mg of gallic acid equivalents / g of extract for MLQ and MFQ respectively.
Flavonoids are the most numerous phenolic compound group. In particular, a close correlation has been suggested between dietary flavonoid intake and decreased mortality from coronary heart disease, partly due to the inhibition of low density lipoprotein (LDL) oxidation and reduced platelet agreeability by flavonoids. Flavonoids have free radical scavenging activity due to inhibition of lipid peroxidation, chelation of iron and copper ions 104. It is now widely accepted that dietary polyphenolic play an important role in protecting the body against chronic diseases, such as cancer, cardiovascular diseases and diabetes mellitus105. Flavonoids present in diet may give relief from viral infection, inflammation, allergic reaction, and oxidative stress. As multiple benefits of eating flavonoid rich plant foods for human health are well documented, increasing particular bioactive flavonoid species in plant foods has become of great interest106, 107. The total flavonoids content of EA soluble fraction of leaves and flowers were found to have 0.0061 and 0.008 mg of rutin equivalents / g of extract respectively.
This Essay is
a Student's Work
This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.Examples of our work
Diabetes mellitus ranks highly among the top ten disorders which cause mortality throughout the world. Co-morbidities such as obesity and hypertension due to rise in lipid level may be associated with diabetes. Diabetes mellitus being chronic disorder, treatment without side effect for long term control is important. Present antidiabetic agent possess side effect as risk of hypoglycemia, anemia and cholestatic jaundice107. There has been growing public interest in herbal medication for treatment of diabetes.
Oxidative stress implicated in the pathology of many diseases and conditions including diabetes, cardiovascular diseases, inflammatory conditions, cancer and ageing. Antioxidants may offer resistance against the oxidative stress by scavenging free radicals inhibiting lipid peroxidation108, 109.
Quisqualis indica Linn (MLQ and MFQ) is used in various diseases and disorders for centuries in focal medicine systems110. The derangement of glucose, fat and protein metabolism during diabetes, results into development of hyperlipidemia111. Traditionally Quisqualis indica Linn extracts are used in the treatment of diabetes and lipid disorder. Phytochemicals such as flavonoids, alkaloids, terpenoids, tannins and saponins are frequently implicated as having antidiabetic effects112, 113. Quercetin isolated from Quisqualis indica Linn has been already reported as inhibitor of aldose reductase enzyme, which is responsible for conversion of glucose to sorbitol. Accumulation of sorbitol in diabetes was reported to be responsible for the diabetic complications such as retinopathy, nephropathy. The formation of inflammatory mediators such as cyclo-oxygenase and lipo-oxygenase were reported to be inhibited by quercetin114,115. Thus from above reported activities of Quercetin, phytoconstituents of Quisqualis indica Linn may be beneficial in the management of diabetes116, 117.
Rutin also showed promising activity in diabetes, it decreases the levels of blood glucose and HbA1c in patients with diabetes mellitus118. Terpenoids and tannins are the major chemical constituents of Quisqualis indica Linn13. On the basis of traditional claims, methanolic extract of leaves and flowers of Quisqualis indica Linn extracts (MLQ and MFQ) were chosen for this study to assess its efficacy in the treatment of diabetes and its complications.
In the hypoglycemic study, oral administration of MLQ and MFQ at all doses did not show any significant change in blood glucose in normoglycemic animals. Also group treated with glimepride unable to affect blood glucose level when compared with control.
In the antihyperglycemic study it was observed that, in the control group 30 min after the glucose administration the blood glucose concentration increased rapidly and then decreased slowly. Administration of glimepride into the animal prior to the glucose loading shows time dependant and statistically significant lowering of blood glucose. There was an improvement in glucose tolerance after administration of extracts MLQ and MFQ. All doses of extracts MLQ and MFQ showed significant antihyperglycemic activity upto 150 min as compared to control. MLQ and MFQ produced dose dependant antihyperglycemic activity. The antihyperglycemic activity of extracts MLQ and MFQ in glucose loaded animals may be mediated by increase in insulin release from pancreas or may be due to increased insulin sensitivity. This activity may be because of inhibition of intestinal absorption of glucose or by stimulating glucose uptake in the peripheral tissues. When insulin binds to its receptors glucose transporters are recruited towards the plasma membrane which are responsible for glucose uptake50.
Exposure to glucocorticoids in large amounts, as occurs during glucocorticoid treatment, Cushing's syndrome and mental stress causes insulin resistance. Insulin resistance occurs in skeletal muscles, adipose tissues and in liver. In skeletal muscle and adipocyets, insulin stimulates glucose uptake via translocation of GLUT-4 from intracellular vesicles to cell membrane. Intracellular lipid induced inhibition of insulin receptor substrate (IRS)-1 tyrosine phosphorylation results in reduced phosphatidyl inositiol 3 kinase (PI-3K) activity which decreases insulin signaling leading to insulin resistance114, 115.
Dexamethasone increases plasma glucose and triglyceride levels, causing an imbalance in carbohydrates, proteins and lipid metabolism leading to hyperglycemia and hyperlipidemia. Glucocorticoids may increase circulating free fatty acids by activating lipoprotein lipase. Increasing free fatty acids compete with pyruvate for mitochondrial oxidative metabolism, ultimately leading to development of triglyceride level in muscles as well as other tissues, which are then deposited in these organs119.
In the present study, dexamethasone administration resulted in increased blood glucose and triglyceride levels. At dose of 400 mg/kg, p.o. of methanolic extracts of leaves and flowers of QI prevented the rise in blood glucose and triglyceride levels caused by dexamethasone. Extracts MLQ and MFQ (200 and 400 mg/kg, p.o.) also caused significant increase in glucose uptake in mice isolated hemidiaphragms which might be due to increase in the insulin sensitivity.
Glucocorticoid treatment is known to induce insulin resistance and catabolic states in rats120. Pharmacological doses of glucocorticoids induce ob gene expression in rat adipocytes within 24 h, which is followed by complex metabolic changes like increased in leptin level, resulting in decrease in food consumption, reduction in body weight with enhanced blood glucose and triglyceride levels121,122. As expected in the present study dexamethasone induced catabolic state and showed reduction in body weight, while MLQ, MFQ and pioglitazone treatment inhibited dexamethasone-induced reduction in body weight and showed significant increase in body weight.
It is generally accepted that sequence of events leading to hepatocytes fatty degeneration begins with insulin resistance and excess of intracellular fatty acids, oxidative stress, and mitochondrial dysfunction leads to injury to hepatocytes. Lipid retention within hepatocytes due to insulin resistance triggers oxidative stress at different levels. In particular, the alteration of intracellular fatty acid trafficking and mitochondrial beta oxidation, consequent to diperlipin, adipolipin leading to impairment of hepatic turnover and leads to hepatic accumulation of lipids and further to peroxidation of lipid molecules and elevates malondialdehyde level123,124.
Uncoupling proteins (UCP) play role in diabetes, catalyze the inducible proton conductance and disperse proton electrochemical potential gradient across mitochondrial inner membrane which results in substrate oxidation and dissipation of oxidative energy125. Members of UCP family are distributed in various tissues (UCP-1) and skeletal muscles (UCP-3). UCP-1 which is expressed in brown adipose tissue is important for controlling dissipation of oxidation energy and it is found to be beneficial in diabetes by reducing excessive oxidative energy. UCP-3 responsible for glucose homeostasis, facilitate fatty acid oxidation and decrease ROS production126,127.
Methanolic extracts of leaves and flowers of QI showed significant increase in various antioxidant enzymes which perhaps via increasing level of UCP-1 and UCP-3. MLQ and MFQ also stimulated skeletal muscle glucose uptake which might be due to prevention of uncoupling reaction in the mitochondria. MLQ and MFQ showed significant increase in antioxidant enzymes levels GSH, SOD, Catalase and decrease in LPO which protect the tissues from ROS induced damage.
Alloxan is commonly used to produce diabetes mellitus in experimental animals due to its ability to destroy the Î²-cells of pancreas possibly by generating excess reactive oxygen species such as H2O2, Oâ€¢2 and HOâ€¢, which leads to persistent hyperglycemia. Reactive oxygen species play an important role in the etiology and pathogenesis of diabetes mellitus and its complications because of generation of oxidative stress128. Alloxan induced diabetes in experimental animal shows structural and functional changes in liver and kidney similar to the once observed in severe diabetes mellitus. Alloxan induced rats produce cardinal signs of diabetes mellitus such as polydypsia, polyphagia, increase in blood pressure, decrease in heart rate and loss of body weight129,130.
In the present investigation diabetes was induced by a single intraperitoneally injection of alloxan monohydrate in citrate buffer (pH 4.5) at a dose of 140 mg/kg body weight of the rat. The diabetic state was confirmed 48 hr after alloxan injection by hyperglycemia. Surviving rats with fasting blood glucose level higher than 250 mg/dl were included in the study.
In the present investigation, rats in the diabetic control group had characteristic hyperglycemia, hyperlipidemia and decreased HDL-cholesterol level when compared with normal control. The plasma glucose on day 41 revealed that MLQ and MFQ produced significant antihyperglycemic activity.
In the present investigation, the antihyperglycemic activity of MLQ and MFQ in alloxan-induced diabetic animals could possibly due to inhibition of intestinal absorption of glucose or by suppressing enzymes involved in gluconeogenesis or by stimulating glucose uptake in the peripheral tissues or due to increased insulin sensitivity.
Insulin is not only involved in regulation of carbohydrate metabolism but also important role in the metabolism of lipids. Chronic diabetes is always associated with derangement in lipid metabolism. Increased levels of free fatty acids into the circulation were because of enhanced activity of hormone sensitive lipases in diabetes. Excess levels of acetyl-CoA, and cholesterol in diabetes was due to the increases in the Î²-oxidation of fatty acids131. Since insulin inhibits the activity of the hormone sensitive lipases in adipose tissue and suppresses the release of free fatty acids, it is potent inhibitor of lipolysis. Insulin has an inhibitory action on HMG-CoA reductase, a key enzyme that acts as a rate limiting enzyme in the metabolism of LDL-cholesterol132. In Diabetes, clearance of LDL-cholesterol by insulin was impaired which leads to the hypercholesterolemia. Diabetes-induced hyperlipidemia is attributed to excess mobilization of fat from adipose owing to less utilization of glucose133. In preclinical experimental diabetes hypercholesterolemia and hypertriglyceridemia have been reported. Decrease in the membrane fluidity was reported in diabetic condition due to hypercholesterolemia which is responsible for relative molecular ordering of the residual phospholipids. In diabetes, level of HDL is decreased and as a result of decrease in level of HDL chances of heart disease increases134,135.
Coronary heart disease was may be owing to excessive accumulation of triglycerides in body. In the present investigation the level of triglyceride in diabetic control rats showed significant increase. MLQ and MFQ significantly reduced the triglyceride level in alloxan-induced diabetic rats. Diabetic animal treated with MLQ and MFQ, glimepride and losartan showed significant decrease in the triglyceride level possible mechanism for triglyceride lowering activity of MLQ and MFQ may be either because of increase in uptake and utilization of glucose leading to subsequent inhibition of lipolysis. In the present study, all diabetic MLQ and MFQ treated groups showed significant and dose dependant reduction in levels of plasma cholesterol and LDL-cholesterol, and improvement in HDL-cholesterol levels, which may be caused by decrease in lipolysis by direct action of MLQ and MFQ on lipoprotein lipase and increase in removal of LDL so, this activity also may have played role in hypocholesteremic activity of MLQ and MFQ.
Insulin is the main regulator of glycogenesis in muscle and liver. There is decrease in the hepatic and skeletal muscle glycogen content in diabetic rats136.
Loss of body weight could be due to dehydration and catabolism of fats and protein seen during diabetes mellitus138,139. It is reported that the recovery in body weight is far less in the poorly controlled diabetic rats as compared to well-controlled diabetic rats. In the present study diabetic control group rats showed significant loss of body weight. All animals treated with MLQ and MFQ showed significant prevention of the loss in body weight throughout the study. This prevention of loss in body weight by MLQ and MFQ is may be due to increase glucose uptake in peripheral tissues or inhibiting catabolism of fat and protein or by glycemic control.
Diabetic cardiomyopathy is one of the important macrovascular complications which may be responsible for the heart failure in various diabetic patients. Clinically it has been reported that persistent hyperglycemia, one of the major cause of the development of unrelieved diabetic complications. In the pathogenesis of diabetic complications such as cardiomyopathy involves myocytes necrosis, vascular endothelial cell dysfunction, and formation of advanced glycation end products (AGE)140.
Sustained hyperglycemia causes vascular endothelial cell dysfunction, resulting in increased permeability, reduced blood flow, and subsequently tissue ischemia. In response to tissue ischemia, endothelial cells release growth factors that increase basement membrane (BM) thickening and extracellular matrix (ECM) deposition141.
Diabetes mellitus and hypertension are common chronic conditions that frequently co-exist. Approximately 80% of the diabetic patients are hypertensive whereas 5-25% of hypertensive individuals are diabetic. Arterial hypertension is twice as common in both Type-I and Type-II diabetes as compared to general population142. Numbers of factors are involved in the pathogenesis of hypertension in diabetes mellitus such as sodium retention, extracellular fluid volume expansion, altered activity of sympathetic nervous system and renin angiotensin system (RAS) and increased vascular reactivity towards noradrenaline and angiotensin-II, and these changes may contribute to the disorders of cardiovascular regulation including blood pressure142.
Cardiac depression in the diabetic state could be a result of microangiopathic changes, altered cardiac autonomic functions143, increased stiffness of ventricular wall144 and changes in sub cellular organelles such as sarcoplasmic reticulum145, membrane pumps and enzymes in the sarcolemma146. The decrease in the heart rate could be attributed to a down regulation of myocardial beta-receptors, increase in levels of circulating cardiac catecholamines. There is decrease in beta-receptor binding sites in the diabetic heart, along with altered myocardial calcium metabolism and reduced uptake of calcium by the sarcoplasmic reticulum and concomitantly depression of SR-calcium ATPase activity147.
In the present study it was observed that treatment with MLQ and MFQ significantly protected the diabetic animals from elevation of blood pressure. Diabetic rats treated with glimepride showed less significant reduction in blood pressure but there was increase in heart rate which may be due to inactivation of K+ channel and increase entry of Ca++ in myocardium tissue148.
Diabetic nephropathy (DNP) is a major cause of illness and premature death in diabetic patients, largely through accompanying cardiovascular diseases and end-stage renal failure149. Diabetes induced by alloxan in rats results in development of nephropathy similar to early stage clinical diabetic nephropathy150. Diabetes produces qualitative and quantitative changes in the composition of the basement membrane and this altered material undergoes accelerated glycosylation and further rearrangement to form advanced glycation end-products (AGEs), which stimulate protein synthesis, further decrease degradability of the basement membrane, increase its permeability and cause endothelial dysfunction. Hyperglycemia increases the expression of transforming growth factor Î² in the glomeruli and of matrix protein specifically stimulated by cytokine. MLQ and MFQ may contribute to the both cellular hypertrophy and enhanced collagen synthesis is observed in diabetic nephropathy151.
During diabetes, there is increased protein catabolism with inflow of amino acids to liver, which feed gluconeogenesis and accelerate ureagenesis, resulting in hypoproteinemia and hypoalbuminemia138,152. The diabetic hyperglycemia induces elevations of the levels of plasma creatinine, urine total protein and urine albumin which are considered as significant markers of renal dysfunction153.
In the present study diabetic animals treated with MLQ and MFQ showed reduction in in the plasma total protein and albumin levels. Treatment with MLQ and MFQ also prevented the rise in plasma creatinine level. These results indicate that MLQ and MFQ attenuate the progression of renal damage in alloxan induced diabetic rats. Effect of MLQ and MFQ on plasma total protein, creatinine and albumin showed that, MLQ and MFQ decreases the progression of diabetic nephropathy in alloxan diabetic rats.
Excessive production and accumulation of lipids can have devastating effect on renal structure and function152,153. The antihyperlipidaemic property of MLQ and MFQ may also have contributed to its beneficial effect on diabetic nephropathy. The use of typical antioxidants alone or in combination may retard or even prevent the normal progression of diabetic complications153.
In addition to hyperglycemia, glycation of proteins are associated with the development of diabetic complications, resulting in generation of free radicals154. Hence, the antioxidant effect along with the glycemic control exerted by MLQ and MFQ may have mediated the protective effect against the nephropathy and cardiovascular complications in alloxan induced diabetic animals.
Hence, the results obtained in the present study indicate that extracts of MLQ and MFQ have the potential to treat diabetes mellitus and prevent diabetes mellitus associated nephropathy and cardiovascular complications.
Inflammation begins when pro-inflammatory substances such as cytokines, thrombin, or reactive oxygen species (ROS) which are released in response to various tissue disturbances. These factors activate several cell types participating in coordinate response. At the initial step, activated platelets and/or polymorphonuclear (PMN) leukocytes (Neutrophils and Eosinophils) adhere to the vessel wall at the site of disturbance. Subsequent PMN filtration through the endothelial barrier allows these cells to target pathogenic source in affected tissue. This process is followed by the transmigration of monocytes and their differentiation into macrophages, which, serve to clean the site of disturbance from the damaged cell debris155,156. Arachidonic acid metabolites are known to contribute to both the development and resolution of inflammation, which makes temporal and compartmental regulation of eicosanoids synthesis and release critical to the inflammatory process. Because eicosanoids are primarily short-lived biomolecules which act in close proximity to the site of synthesis, tissue specific responses depend upon which arachidonic acid products are generated at the scene of interest and which receptors are present in the responding cells157. Reactive oxygen species cause induction of various cytokines via activation of transcription factors like NF-kB and activated protein (AP-1)156,158,159. Oxidants also initiate production of inflammatory mediators like IL-8, upregulation of cyclo-oxygenase160. The inflammatory process involves a series of events which can be elicited by numerous stimuli such as infectious agents, ischemia, and disturbance in arachidonic acid pathway leading to expression of COX-II at cellular level and thermal and physical injury. Inflammation is also associated with secondary processes resulting from the release of analgesic mediators160.
Various in-vivo and in-vitro methods are developed for the evaluation of anti-inflammatory agents. However, among the in-vivo methods the carrageenan induced rat paw edema assay is believed to be most reliable and widely used animal model for anti-inflammatory compounds.
Carrageenan used as an edemogen was introduced by Winter et.al. in 1962. Carrageenan induced paw edema was taken as a prototype of exudative phase of inflammation. Carrageenan induced inflammation is useful model for detecting the orally active anti-inflammatory agents. Carrageenan in the rat paw edema is known for its biphasic response. The first phase is due to release of histamine, serotonin and kinins during one to two hours after injection of carrageenan, while second phase of edema from three to six hours which may be due to the discharge of prostaglandins, protease and lysosomal enzymes161,162,163. The second phase is most responsive to the majority of anti-inflammatory drugs164. Plasma extravasation, increased tissue water and plasma protein exudation along with neutrophils extravasation, caused by metabolism of arachidonic acid was observed after sub-planter injection of carrageenan in to the paw of rat165.
In the present study, it was observed that methanolic and pet ether extracts of Quisqualis indica Linn showed significant decrease in paw edema volume from 1st to 12th hr when compared to control group suggesting the mechanism action may be mediated by inhibition of inflammatory mediators like prostaglandin, cytokines and chemokines.
It is well known that inhibition of formaldehyde-induced paw edema in rats is one of the most suitable test models to screen anti-arthritic and anti-inflammatory agents as it closely resembles the human arthritis164,166. An admirable chronic inflammatory experimental model in which leukocyte migration, fluid extravasation and biochemical exudates observed in inflammatory response can be easily noticed in cotton pellet-induced granuloma168. Angiogenesis, nitric oxide synthesis and kinins release are major causes of the granuloma. Facilitation of migration of inflammatory cells to the site of inflammation along with supplies of nutrients and oxygen was observed in angiogenesis. Therefore, the suppression of angiogenesis is important to inhibit the development of chronic granulation tissue135. Many mediators found to be involved in the formation of the granuloma, such as cytokines169, chemokines170 and eicosanoids171. The dry weight of the pellets in this model correlates well with the amount of granulomatous tissue. Proliferation of neutrophils, macrophages, fibroblasts and development of small vessels, which are the basic sources of forming a highly vascularised reddish mass termed "granulation mass" was observed at the time of repairing of inflammation168.
The anti-inflammatory activity of flowers and leaves of Quisqualis indica Linn was evaluated in chronic model of inflammation i.e. cotton pellet induced granuloma in mice which represents the proliferative phase of inflammation involving tissue degeneration and fibrosis.
Methanolic and pet ether extracts of Quisqualis indica Linn was found to reduce granuloma mass dose dependently by inhibiting the proliferative phase of inflammation in cotton pellet granuloma model in mice indicating chronic anti-inflammatory action.
Treatment with methanolic and pet ether extracts of Quisqualis indica Linn significantly restored the level of non-enzymatic antioxidant GSH and enzymatic antioxidant enzymes including Catalase and SOD in cotton pellet induced granuloma indicating its role as an antioxidant in chronic stage of inflammation. This antioxidant activity may be due to presence of flavonoids.
Thus from the results obtained from present experiment it can be concluded that the extracts of Quisqualis indica Linn significantly decrease the LPO level and prevented the cell damage by reducing oxidative stress.
In cotton pellet induced granuloma, extracts of Quisqualis indica Linn did not show any significant sign of ulcer in cotton pellet treated mice. Thus ulcerogenic study revealed that, extract has no adverse effect on the gastric mucosa.
Despite the different chemical structure of aspirin-like drugs, the analgesic effect of NSAID's is mainly due to the inhibition of the cyclooxygenase enzyme which is involved in the formation of prostaglandins. NSAID's relieves pain by normalization of the increased pain threshold associated with inflammation via inhibition of the formation of prostaglandins. Much of the research into the roles of the COX enzymes in pain has focused on the action of the key downstream prostanoid, PGE2. The mechanisms by which PGE2 modulates the pain pathway are complex with pleiotropic actions likely to synergize to mediate the eventual biological effects. Four receptors, coupled to different signal transduction pathways, are identified for PGE2: EP1, EP2, EP3 and EP4 and are believed to mediate the diversity of actions of PGE2172,173. The EP1 receptor is coupled to intracellular Ca2+ mobilization, whereas EP2 and EP4 are coupled to stimulation of adenylate cyclase via GS while EP3 is coupled to inhibition of Adenylate Cyclase via GI receptor pathway. The relative contribution of each of these receptors is far from clear, and the conflicting literature cites all of them as essential, in some way, in pain or inflammation174,175,176.
Acetic acid induces pain by enhancing levels of PGE2 and PGF2Î±177 at the receptors of peritoneal cavity177,178,179 which suggested that acetic acid acts indirectly by increasing the release of endogenous mediators, leads to stimulation of the nociceptive neurons which are sensitive to most of the non-steroidal anti-inflammatory drugs and narcotics agents. Acetic acid is a model of visceral pain causes irritation to peritoneum, which induces writhing activities180,181.
Increased vascular permeability and oedema are among the earliest events in the inflammatory response followed by infiltration of leukocytes. These events are initiated and maintained by a sequence of inflammatory mediators of cellular and plasma origin182. This process involves vascular and cellular occurrences in response to tissue injury against physical, chemical or infective aggressions182. Due to these vascular and cellular events, the neutrophils migrates from blood into affected tissues and involves diverse mediators such as pro-inflammatory cytokines (TNF-Î±, IL-1, IL-12), chemokines, eicosanoids, leukotriene and C5A release from resident cells. These factors later induces rolling and adhesion of neutrophils on endothelial cells, followed by their transmigration to the extra vascular space mediated by adhesion molecules183. Acetic acid increases the prostaglandin E2 (PGE2), prostaglandin F2Î± (PGF2Î±), serotonin, and histamine levels in the peritoneal fluid, which results in dilation of the capillary vessels and increase in vascular permeability, finally proteins are extraverted in extracellular fluid and leads to edema formation183. It is therefore used as a well-characterized mouse model of acute inflammation, and inhibition of vascular permeability is considered a major feature for the suppression of the exudative phase of acute inflammation. Vascular permeability induced by acetic acid was measured by Evan's blue dye from the abdominal cavity. Evan's blue binds to plasma proteins and extravasates with them at sites of increased permeability. The increases of vascular permeability are caused by a leak of plasma from the blood vessels in inflammatory swelling. This leads to the dilation of arterioles and venules which leads to vascular permeability184.
Methanolic and pet ether extracts of Quisqualis indica Linn may inhibits release of the prostaglandin E2 (PGE2), prostaglandin F2Î± (PGF2Î±), serotonin, and histamine, which inhibits dilation of the capillary vessels and decreased in vascular permeability in this model.
For establishing analgesic activity, methanolic and pet ether extracts of Quisqualis indica Linn were studied for acetic acid induced writhing method. Acetic acid produces a painful reaction and acute inflammation in the peritoneal area. Acetic acid induced abdominal writhing is visceral pain model. Acetic acid releases arachidonic acid via cyclooxygenase and prostaglandin biosynthesis which plays a key role in the nociceptive mechanism185. It induces pain by enhancing fluids of PGE2 and PGF2Î± at the receptors of peritoneal cavity186,187, which suggested that acetic acid acts indirectly by increasing the release of endogenous mediators and leads to stimulation of the nociceptive neurons which are sensitive to most of the non-steroidal anti-inflammatory drugs and narcotics agents188. The reduction of writhing responses is probably related to the reduced synthesis of the inflammatory mediators by inhibition of cyclo-oxygenase and/or lipoxygenases189. In present investigation, extracts of Quisqualis indica Linn inhibits writhing in dose dependant manner which suggested the involvement of peripheral mechanisms of analgesia. The analgesic effect could be due to the inhibition of release of endogenous inflammatory mediators, which can be correlated with the results obtained in the carrageenan induced paw edema model, where it had shown the inhibition of paw edema from 1st to 12th hr, indicating the inhibition of inflammatory mediators like prostaglandin, cytokines and chemokines .
Hot plate method is supraspinally mediated and has selectivity for centrally acting analgesics190,191. In this method increase in the reaction time is considered for evaluating central anti nociceptive activity192. This method is used to differentiate between central and peripheral analgesics. The centrally acting analgesics increase the reaction time in the hot plate model190.
The extracts of Quisqualis indica Linn exhibit the increase in reaction time in hot plate model which clearly indicates that the extracts of Quisqualis indica Linn may act as a central analgesic.
Formaldehyde-induced arthritis is a model used for the evaluation of an agent with anti-proliferative activity167. Injection of formaldehyde into subcutaneous region in rats produces significant local inflammation.
The most important transmission pathways for inflammatory pain are ion-sensitive peripheral polymodal nociceptors (Acid sensitive ion channel, Vanilloid receptor-1, and Glutamate receptors) and algogenic substances (Bradykinin, Prostaglandin and Cytokines as Tumor necrosis factor-Î±, Interleukin-1Î², Interleukin-8)192. Our data indicate that the antinociceptive action of the extract in the chemical pain models (i.e. acetic acid-induced writhing and formalin-induced licking) could be due to inhibition of cytokine release or antagonism of acid sensitive ion channels, vanilloid and /or glutamate receptors. It follows that decreased levels of cytokines and lipid mediators in the peritoneal cavity may be the reason for the reduction of cell migration. Furthermore, reduction of vascular permeability could be associated with reduced levels of prostaglandins and leukotrienes, as both are inflammatory mediators related to vasodilatation192. Extracts of Quisqualis indica Linn inhibited second phase of formalin induced nociception in mice, indicating analgesic effect which was probably by inhibiting the release of inflammatory mediators.
From present study, it can be concluded that the extracts of Quisqualis indica Linn has anti-inflammatory and analgesic activity which might be due to the inhibition of inflammatory mediators like prostaglandin, cytokines and chemokines.