WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 7.632 Volume 10, Issue 8, 2666-2738 Research Article ISSN 2278 – 4357
PRELIMINARY PHYTOCHEMICAL AND ANTIDIABETIC EVALUATION OF ORTHIOSIPHION THYMIFLORENS IN DIABETIC INDUCED WISTAR RATS
Mariyam Begum*
Associate Professor in Shadan Women's College of Pharmacy. Khertabad, Hyderabad.
1. INTRODUCTION Article Received on 21 June 2021, 1.1. Diabetes mellitus
Revised on 11 July 2021, Diabetes mellitus is a heterogeneous metabolic disorder relating to Accepted on 01 August 2021 [1] DOI: 10.20959/wjpps20218-19798 carbohydrate, fat and protein metabolism. Deficiency or insensitivity of insulin causes glucose to accumulate in the blood, called as hyperglycemia. This leads to the polydipsia, polyuria, and *Corresponding Author polyphagia and weight loss.[2] Mariyam Begum Associate Professor in 1.1.1. Epidemiology Shadan Women's College of Pharmacy. Khertabad, Diabetes mellitus is a verdure problem involving millions of
Hyderabad. individuals in the area. World Health Organization expected that 300 millions of people will suffer with diabetes mellitus by the year 2025.[3]
From this 80% would be in developing countries. It is more a fatal disorder than AIDS at present killing a great number of people.[4]
1.1.2. Types of diabetes Diabetes mellitus is of two types. Type 1 (insulin based or juvenile onset diabetes) Type 2 (Non insulin dependent or maturity onset diabetes) Type 1: In this type of diabetes mellitus destruction of ß cells leads to the lack of insulin secretion.
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Patients are usually young (children or adolescents) Causes 1. Genetic susceptibility 2. Autoimmune factors 3. Environmental factors (infections) Type 2: It is coupled with insulin resistance and impaired insulin secretion. Patients are often obese and the incidence rises progressively with age as ß cell function declines.
Causes 1. Genetic factors (80%) 2. Constitutional factors (obesity, hypertension, decreased physical activity). [ 5 ]
Diabetes mellitus is combined with debilitated glucose metabolism that directs to an increase in free radical production, increase in triglyceride, lipoprotein levels. Free oxygen radical should start oxidation of lipids, which is responsible for the titillation of glycation of protein, inhibition of antioxidant enzymes and take part a role in the long term complications of diabetes.[6] Uncontrolled diabetes leads to several microvascular (Nephropathy, Neuropathy and Retinopathy) and macrovascular (Atheroma) complications and have an effect on many organs of the body.[7]
1.2. Diabetic nephropathy The kidneys opera a decisive role in conserving overall health. These couple organs filter waste products and extra water from the blood and also involve in synthesis of hormones that are chief for the best maintenance of body functions. Increased blood glucose in diabetes leads to damage of many small blood vessels present in kidneys. Overtime, the kidneys are not adequate to work entirely and this edge to kidney failure.
It is a continuous and irreversible renal disease characterized by the deposition of extracellular matrix in glomerular mesangium and kidney interstitial tissue that leads to renal failure.[8]
1.2.1. Epidemiology DN affects approximately 30-35% people suffering from type 1 or type 2 DM.[9] Type 2 diabetes is a main cause to kidney disease in 20-40% of population. 50% will have
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microalbuminuria secondary to hypertension.10-20% with microalbuminuria will progress to overt Nephropathy.[10]
1.2.2. Pathogenesis Several mechanisms are considered to be ramified in the pathogenesis of DN and its obstacles, all of them originating from hyperglycemia and dyslipidemia.[8,11] Metabolic and haemodynamic interactions onward with glomerular proteins glycosylation are to be involved in the pathological process of DN.[12]
Hemodynamic factors that are an addition to the development of DN include elevated systemic and intraglomerular pressure, proactivation of vasoactive hormone alley including the RAAS and endothelin. These alleys turn on the intracellular second messengers that are protein kinase C (PKC), mitogen activated protein kinase (MAP kinase), and nuclear transcription factors (NF), vascular endothelial growth factor (VEGF), cytokines and transforming growth factor (TGF-ß).
Hyperglycemia stimulates PKC through denovo generation of diacylglycerol. Activation of PKC in the glomeruli has been connected with processes elevating mesangial expansion, increasing of thickness of basement membrane, smooth muscle contraction, endothelial dysfunction.
Glucose based pathways are also activated within the diabetic kidney which augment the oxidative stress, free radicals generation and further activate renal polyol pathway causing increase in advanced glycation end products (AGES). In grouping, these routes finally direct to improved renal albumin permeability and extracellular matrix accumulation, ensuing in an increase in proteinuria, glomerulosclerosis and eventually development of tubulointerstitial fibrosis.
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Fig. 1.1: Pathogenesis of diabetic nephropathy.
Oxidative stress is significantly increased in diabetes because persistent hyperglycemia increases the production of free radicals. There is a data that diabetes mellitus increases number of pathways for the increased production of ROS. These included increased glucose oxidation, increased mitochondrial superoxide production, stimulation of NAD(P)H oxidase by angiotensin II (protein kinase c dependent), glucose and advanced glycation end products. The most known free radicals involving in the diabetic nephropathy pathogenesis are ROSuch as superoxide(O2 ), hydroxyl (OH ) and peroxyl (RO2) and non radical species such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) and reactive nitrogen species produced from similar pathways.[13]
Fig. 1.2. Oxidative stress mechanism in diabetic nephropathy. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2669
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Modern studies have recommended diabetic nephropathy as an inflammatory process, and immune cells could be involved in the maturity of diabetic nephropathy. AGES increase inflammation and accelerate the formation of ROS during normal aging, as well as in diabetic patients. Cyclo-oxygenase-2 (COX-2), which shows anti inflammatory properties via inhibition of its enzymatic activity, is increased in diabetic nephropathy.
Stages of DN 1. Hyper filtration, early renal hypertrophy. 2. Glomerular lesions without clinically evident disease. 3. Incipient nephropathy with micro-alb/cr ratio 0.03-0.3 or albumin 20-200µg/min on timed Specimen. 4. Overt diabetic nephropathy with proteinuria > 500mg/24hr, creatinine clearance <70 ml/min. 5. ESRD, Creatinine clearance <15ml/min.
Traditional medicine World Health Organization (WHO) offers the collective term Traditional medicine (TM) to refer systems of medicine such as traditional Chinese, Indian Ayurveda, Arabic Unani and various systems of indigenous medicine. TM is also referred as Complementary or Alternative or Non Conventional Medicine 14 (WHO, 2002).
Advantages of traditional medicine (TM) Easy availability, accessibility, affordability though quality and mechanism differ in various parts of the World. Many of the developing countries are on the increased consumption due to limited availability and accessibility of Modern medicine which is expensive and often unaffordable. Available at the local level and easily affordable 15 (Zhang, 2004). Adds the advantage that it has no drug interventions. It has right quality of material and apt processes are used from sourcing to marketing. No contamination, adulteration or spiking 16(Kaened, 1999). Emerging diseases where no medicines are available have provoked the interest in medicinal plants as a significant source of new medicine 17(Dhaunkar et al., 2000). www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2670
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Cost effective and has limited systematic studies to compare. Contain several chemicals substances, its medicinal action are due to the co-operation of the constituents. Possess natural affinity to the body system, non specific, normalizing action shows minimal disturbances in cellular environment, minimal psychological changes. Recent studies supported the general belief of TM as affordable over modern medicine 18 (Ernest, 2000; Evans, 1997).
Systematic Pharmacognostical, detailed Phytochemical investigations and innovative Pharmacological studies provide scientific information on the quality, efficacy, safety or toxicity of traditional drugs of plant origin which will enhance the standardization and acceptance of Traditional medicine globally.
1.5. Aminoguanidine 1.5.1. Chemistry of Aminoguanidine Aminoguanidine is a nucleophilic hydrazine derivative and small molecular size compound (MW around 74.1 Da).[19] Aminoguanidine is an agent structurally similar to laevo- arginine.
Two main varieties of aminoguanidine are there, the bicarbonate and hydrochloride variety. Even though the bicarbonate compound is more normally presented, the hydrochloride report is supposed to be the most active (bioavailable) as it is more soluble.
1.5.2. Mechanism of action of aminoguanidine Aminoguanidine contains a terminal amino group, which has higher chemical reactivity than terminal amino groups of proteins. Based on this characteristic feature aminoguanidine has been selected as an inhibitor of glucose binding to proteins. Aminoguanidine has been found to reduce the development of AGE products, principally by reacting with Amadori products, i.e. by blocking the carbonyl groups on ketoamines and their derivatives.[20] Aminoguanidine is active mainly against certain aldehyde, which www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2671
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contributes to cross-linking as malondialdehyde and alpha-oxoaldehyde. However, its primary mechanism of action is now believed to be trapping of bicarbonyl intermediates.[21]
1.5.3. Pharmacokinetics of aminoguanidine Aminoguanidine is absorbed after intraperitonial (i.p) administration, peaked in plasma (9μg/ml) at 0.5 hours and had a half life of 1.88 hours. Steady-state values for the area under the curve for therapeutic regimens were 20.5 and 16.35 μg/ hr/ ml.[22]
1.5.4. Pharmacological actions of aminoguanidine Aminoguanidine has aroused an enormous deal of curiosity from the last few years, due to its established ability to obstruct the formation of AGEs and AGEs–produced cross linkages in both preclinical and clinical trial studies. Though it inhibits AGEs formation and AGE- induced cross linkage in collagen and other tissues, it could not impede the development of normal collagen cross- links, which are essential for structural integrity.[23] In experimental diabetic animals, it averts the annoyed-linking in tendons and skin which shows its probable action for prevention of muscle and joint age-related stiffness, and skin aging (wrinkles)[24] It also prevents cardiac enlargement by plummeting the risk of glycation-induced injury to cardiac collagen. Also, it inhibits the cross-linking between lipoproteins, and therefore reduces the threat of obstruction of the arteries.[31] It reduces the development of lipofuscin (age pigment) and prevents diabetic neuropathy and cataract.[25] Moreover, aminoguanidine was able to protect against diabetes-accelerated erectile dysfunction and atherosclerosis.[26,27]
Aminoguanidine is a pathetic copper chelator. Grand levels of free copper are more liable to augment AGE-induced damage. In addition to inhibiting AGE formation, aminoguanidine has other pharmacological activities. It is a selective inhibitor of inducible nitric oxide synthase (iNOS).[28] Furthermore, antioxidant activity of aminoguanidine has been described by some studies. In diabetic subject aminoguanidine has been reported to lower total cholesterol, LDL cholesterol and triglycerides independent of glucose control.[29] It has shown positive actions in civilizing DN in double blind human trials.
1.5.5. Uses of aminoguanidine Aminoguanidine is still under clinical investigation (phase III trial) for the prevention of diabetic complications including neuropathy, nephropathy, and retinopathy. The recommended dose is 300 mg twice a day.[30] www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2672
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Literature survey 2.1. Animal models in experimental diabetes mellitus For the selection of antihyperglycemic agents, DM can be created in animals like mouse, rat, guinea pig, cat, dog, hamster, rhesus monkey etc. Animal models are classified as below Insulin Dependent Diabetes Mellitus (IDDM) analogous animal models. Non-insulin Dependent Diabetes Mellitus (NIDDM) analogous animal models.
2.1.1. IDDM like animal models Destruction of β-cells of langerhns of islets produces the IDDM. This type of diabetes induced by viral infection, injection of diabetogens or by introduction of transgenes. Streptozotocin and alloxan were found to be more selective in β-cells devastation than other diabetogenic agents.
2.1.2. NIDDM like animal models NIDDM animal models can be primed by injecting streptozotocin intravenously at a dose of 100mg/kg to neonatal wistar rats on the day of birth, through sapheneous vein which is easy to get to by transcutaneous puncture.
Another model of NIDDM is by injecting streptozotocin (90mg/kg, I.P) to two days old Sprague dawley rats resulting in temporary hyperglycemia followed by resurgence post prandial hyperglycemia, as well oral glucose intolerance, in the diabetic range is noticeable at 4-6 weeks of age.
NIDDM along with hypertension can be produced by injecting streptozotocin to neonates of the spontaneous hypertensive rats.
2.2. Rodent models for diabetic nephropathy[31] Trustworthy animal models for diabetic nephropathy are a precious tool to understand the molecular mechanisms answerable for this disease and for the preclinical improvement of new therapeutic strategies. Newly a number of genetically modified (knock out and transgenic) mouse strains have been used to afford vital insights in to the roles of oxidative stress, advanced glycation end products, inflammation and profibrotic mechanisms in the progress of diabetic nephropathy. It is dependent on various factors including i) A steadfast technique for establishing a unfailing level of diabetes.
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ii) It should be able to maintain a firm level of diabetes throughout the period of the experiment. iii) Considerate the disease distinctiveness and progression of injury in the rodent strain iv) The attainment of a pathology condition will have clinical significance.
2.2.1. Low-dose mouse model of STZ-induced Diabetic Nephropathy Mice: Male Age: 6-7 weeks B.W: 20-25g Dose of STZ: 10mg/kg I.P
Mouse strain susceptibility to diabetes induced by several low doses of STZ are arranged in the following sequence DBA/2 2.2.2. Moderate and high-dose mouse models of STZ – induced Diabetic Nephropathy Some studies investigative the diabetic nephropathy in mouse strains which are challenging to STZ-induced pancreatic damage have used either a single high dosage of STZ (>(or)=200mg/kg) or a two dose schedule of STZ (2×100-125mg/kg) given on consecutive days. The following procedure describes a two –dose procedure (2×125mg/kg per day STZ) for establishing diabetes in C5TBL/6 mice with lack of genetics which facilitate mild resistant to STZ. Using this procedure, roughly 90% of wild type C57BCL/6 mice will develop evident diabetes within 2 weeks, with a lower frequency expected for more resistant genotypes. 2.3. Rat models of STZ- induced diabetic nephropathy Models of STZ –induced diabetic nephropathy are normally performed in Sprague–dawley (SD), wistar–Kyoto (wky) or spontaneously hypertensive (SHR) rats Sex: Male www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2674 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Age: 8week Weight: 200-250 g Dose of STZ: SD=55 mg/kg, Wky =60mg/kg SHR=45mg/kg Route: I.V. STZ has also been administered intraperitoneally to rats 2.3.1. Uninephrectomized rat model of STZ induced diabetic nephropathy Uninephrectomy is performed in different rat strains (SD, wistar, SHR) which is considered to speed up the evolution of renal damage. Uninephrectomy leads to the swelling of the remaining kidney, which is more enlarged by the development of diabetes. Uninephrectomy causes the increase in glomerular capillary pressure in SHR rats. However, understanding of this model is multifaceted, it is difficult to dissect the virtual assistance of STZ-induced hyperglycemia and uninephrectomy induced changes in glomerular haemodynamics in the development of renal injury Table 2.1: Representative animal models for diabetic nephropathy and their characteristic features (+: very week, +: weak, ++: moderate, and +++: strong).[32] Animal Diabetic Hyperglycemia Albuminuria Renal Histological model type failure features Streptozotocin High dose 1 +++ +++ ++ ++ (mice) Low dose 1 +++ ++ + ++ (mice) Insulin-2 Akita (mice) Males 1 +++ + ++ ++ Females 1 ++ + + + NOD mice 1 +++ +++ + ++ db/db mice 2 + ++ + +++ KK mice 2 ++ ++ - +++ NZO mice 2 ++ ++ - +++ GK rats 2 ++ ++ - +++ Zucker rats 2 ++ + ++ ++ www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2675 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 2.5. Earlier works on composition of polyherbal formulation 1. Eugenia jambolona The work entitled ―Study of hypoglycemic and hypolipidemic activity of Eugenia jambolana pulp and seed extract in streptozotocin induced diabetic albino rats‖ carried out by Bhavana Srivastava.et. al. Published in Asian Journal of Pharmacy and Life science, 2012, vol. 2, issue 1 summarized that seed and pulp extract of Eugenia jambolana has antidiabetic, antihyperlipidemic activity and lowers blood urea in diabetic rats. It also increases body weight of diabetic rats.[33] The research article entitled ―Attenuation of renal dysfunction by antihyperglycemic compound isolated from fruit pulp of Eugenia jambolona in streptozotocin induced diabetic rats‖ by Tanwar et.al. Published in Indian Journal of Biochemistry and Biophysics, 2010, vol. 47 demonstrated significant protective effects of that partially purified compound FIIC on the consequences of hyperglycemia and early stages of experimentally induced diabetic nephropathy.[34] 2. Tinospora cardifolia The research article entitled ―Magnoflorine from Tinospora cordifolia stem inhibits α- glucosidase and is antiglycemic in rats‖ by Mayurkumar et. al. published in Journal of Functional Foods, 2012, vol. 4 concluded that embarrassment of intestinal α-glucosidase enzyme by Magnoflorine offers this plant species as a choice for the treatment of diabetes mellitus and for prevention and control of diabetes.[35] The research article entitled ―Nephroprotector activity of hydroalcoholic extract of Tinospora cardifolia roots on cisplatin induced nephrotoxicity in rats‖ by spandana et.al. Published in Drug Invention Today, 2013, vol. 5. The present investigation suggests that the hydro alcoholic extract of roots of Tinospora cardifolia has protective effect against cisplatin induced nephrotoxicity.[36] The research article entitled ―Prevention and Management of diabetic retinopathy in STZ diabetic rats by Tinospora cardifolia and its molecular mechanisms‖ by Shyam et.al. Published in Food and Chemical Toxicology, 2012, vol.50 demonstrated that Tinospora cardifolia plays a pivotal role in prevention and management of diabetic retinopathy due to its antihyperglycemic, antiangiogenic, anti-inflammatory and antioxidant properties.[37] www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2676 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The work entitled ―Hypoglycemic and other interrelated actions of Tinospora cardifolia roots in alloxan induced diabetic rats‖ by p. Stanely. Et.al. published in Journal of Ethnopharmacology, 2000, vol.70 states that aqueous root extract of T. Cardifolia in alloxan induced diabetic rats caused a considerable reduction in blood glucose and brain lipids.[38] 3. Gymnema sylvestre The research article entitled ―Hypoglycemic effect of Gymnema Sylvestre R.Br leaf in normal and alloxan induced diabetic rats‖ by Sathya et.al. Published in Ansient Science of Life, 2008, vol. 28. This study provided experimental evidence for the herbal plant Gymnema sylvestre in the prevention and curing of alloxan induced diabetic rats without any side effects.[39] 4. Cressa cretica The research article entitled ―Evaluation of Antidiabetic activity of Cressa cretica Linn in alloxan induced diabetes in rats‖by Chaudhary et.al. Published in pharmacologyonline, 2010, vol. 3. Ethanolic extract of Cressa cretica showed significant hypoglycemic effect in Alloxan induced diabetic rat. It also reduced serum cholesterol and increased HDL- cholesterol.[40] 5. Casearia esculenta The research article entitled ―phytochemical and hypoglycemic investigation Casearia esculenta‖ by Chodhury et.al. Published in Journal of Pharmaceutical Sciences, 2006 concluded that different fractions of both isolated and alcoholic and aqueous extracts of esculenta significantly decreased the blood glucose level.[41] 6. Curcuma longa The research article entitled that ―Potential therapeutic effect of Curcuma longa on streptozotocin induced diabetic rats‖ carried out by Azza A.et.al. Published in Global advanced research Journal of Medicine and Medical sciences, 2012, vol. 1, issue 4 concluded that curcumin consist of antioxidant effect that may supply to its protective action beside lipid peroxidation and enhancing effect on cellular antioxidant defense. This activity contributes to the protection against oxidative damage in STZ induced diabetes.[42] www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2677 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The review article entitled ―Renoprotective effect of the antioxidant curcumin: Recent findings‖ by Joyce Trujillo et.al. Published in redox biology, 2013, vol. 1 identified that curcumin as a promising renoprotective molecule against renal injury.[43] 7. Swertia chirata The research article entitled ―Potential hypoglycemic effect of Swertia chirata an Indian subcontinent herb with important medicinal value‖ by Alam et.al. Published in pharmacologyonline, 2011, vol. 2. This study confirmed the use of Swertia chirata in ethnomedical application for diabetes management.[44] 8. Centratherum anthelminticum The research article entitled ―Antidiabetic activity of Centratherum Anthelminticum Kuntze on alloxan induced diabetic rats‖ carried out by Bhatia et.al. Published in pharmacologyonline, 2008, vol. 3 summarized that aqueous extract of Centratherum anthelminticum showed dose dependent percentage of blood glucose reduction in diabetic rats.[45] 9. Picrorrhiza kurroa The research article entitled ―Antidiabetic activity of standardized extract of Picrorrhiza kurroa in rat model of NIDDM‖ carried out by Gulam Mohammed hussain et. al. published in Drug Discov Ther, 2009, vol. 3, issue 3 concluded that extract of Picrorrhiza kurroa has an antihyperglycemic effect. Therefore it may be potentially beneficial in type 2-diabetes and related dyslipidaemia.[46] 10. Trigonella foenum graecum The research article entitled ―Antioxidant activity of Trigonella foenum graecum using various in vitro and exvivo models‖ by N.Subhashini et.al. Published in International Journal of Pharmacy and Pharmaceutical Sciences, 2011, vol. 3, issue 2 indicated that ethanol extract of T. foenum graecum is effective against free radical mediated disease.[47] The work entitled ―Antidiabetic activity of Trigonella foenum graecum L. Seeds extract (IND01) in neonatal streptozotocin-induced (N-STZ) Rats‖ by Chetan P.Kulkarni.et.al. Published in Diabetologica Croatica, 2012, vol. 41, issue 1 concluded that IND01 (100mg/kg, oral) and glyburide (10mg/kg, oral) showed considerable exchange in- STZ- induced changes (rise in SG, turn down in body weight and rise in HBA1C). Histology www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2678 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences sections of pancreas from the rats treated with IND01 (but not glyburide) showed augment in number and size of pancreatic islet ß-cells. IND01 showed a probable improvement in symptoms of DM during progressive worsening and better glycemic functions in n-STZ induced diabetic rats.[48] 11. Terminalia chebula The research article entitled ―Antidiabetic and renoprotective effects of the chloroform extract of Terminalia chebula Retz. Seeds in streptozotocin-induced diabetic rats‖ by Nalamolu Koteswara Rao.et.al. Published in BMC Complementary and Alternative Medicine, 2006, vol. 6, issue 17 concluded that chloroform extract of seeds of T. chebula formed dose-dependent reduction in blood glucose and analogous with that of drug, glibenclamide in short term study & long term study. Considerable nephroprotective activity is observed in T. chebula treated rats.[49] 12. Holarrhena antidysenterica The research article entitled ―Efficacy of aqueous extract of seed of Holarrhena antidysenterica for the management of diabetes in experimental model rat: A correlative study with antihyperlipidemic activity‖ by Ali et.al. Published in International Journal of Applied Research in Natural Products, 2009, vol. 2 issue 2. The results of this study enlightened that the aqueous extract of seeds have antidiabetic and antihyperlipidemic activities.[50] 13. Pterocarpus marsupium The research article entitled ―Antidiabetic activity of heart wood of Pterocarpus marsupium Roxb. and analysis of phytoconstituents‖ carried out by Akansha Mishra.et.al. Published in Indian Journal of Experimental Biology, 2013, vol. 51 concluded that results of this study demonstrate the usefulness of heart wood of P. marsupium for civilizing on the whole glycemic control and thereby sinking the risk of diabetic complications.[51] 14. Glycyrrhiza glabra The research article entitled ―Antidyslipidaemic activity of Glycyrrhiza glabra in high fructose diet induced dyslipidemic Syrian golden hamsters‖ by Santosh kumar Maurya. et.al. Published in Indian Journal of Clinical Biochemistry, 2009, vol. 24, issue 4 indicated that 95% ethanolic extract of root of Glycyrrhiza glabra and its fractions like water soluble, ethyl acetate soluble and hexane soluble showed decrease serum cholesterol. In the other www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2679 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences way ethanolic extract, ethyl acetate soluble, water soluble and hexane soluble portion showed increase serum HDL-C. Ethanolic extract, ethyl acetate fraction, aqueous fraction, hexane part showed decreased triglyceride level. Ethanolic extract, ethyl acetate soluble portion and water soluble fraction showed reduction in LDL- cholesterol.[52] 15. Mineral pitch The research article entitled ―Effect of shilajit on blood glucose and lipid profile in alloxan induced diabetic rats‖ by Trivedi et.al. Published in Indian Journal of Pharmacology, 2004, vol. 36, issue 6 concluded that due to its complex action Shilajit can offer a new and hopeful approach in the long time management of diabetes mellitus.[53] 16. Tribulus terrestris The research article entitled that ―Hypoglycemic effect of saponin from Tribulus terrestris‖ by Li M et.al. Published in Journal of Chinese Medicinal Materials 2002 vol 25 issue 6 concluded that saponin from Tribulus terrestris could significantly decrease the level of serum glucose.[54] 17. Withania somnifera The research article entitled ―Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root by Andallu et.al. Published in Indian Journal of Experimental Biology, 2000, vol. 38. This study suggested that its being source of hypoglycemic, diuretic and hypocholesterolemic agents the root of the Withania somnifera had a definite potential therapeutic value without detrimental side effects in humans.[55] 18. Nordo Stachys jatamansi The research article entitled ―Stress modulating antioxidant effect Nardostachys jatamansi‖ by Nazmun Lyle et.al. Published in Indian Journal of Biochemistry & Biophysics, 2009, vol. 46 summarized that ethanolic extract of Nardostachys jatamansi attenuated stress- induced elevation of biochemical changes such as membrane LPO, elevated NO production in brain as well as stomach and increase in antioxidant enzyme like Catalase, which are consistent with its anti-stress properties. Invitro study showed that it has potent free radical scavenging action.[56] www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2680 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 19. Bacopa monniera The research article entitled ―Antihyperglycemic activity of Bacosine, a triterpene from Bacopa Monniera, in alloxan induced diabetic rats‖ by Thritha Ghosh et.al. Published in Planta Medica, 2011, vol. 77, issue 88 concluded that Bacosine showed significant decrease in blood glucose and attenuated the antioxidants.[57] 3. AIM AND OBJECTIVE We, the human being possess a huge wealth of medicinal plants which have been explored and validated for their therapeutic properties. Still there are so many plants whose medicinal properties are not yet published and lots of research works are needed to be carried out on such medicinal plants. Herbal drugs play a vital role in the management of various liver disorder, most of them speed up the natural healing process of liver. Numerous medicinal plants and their formulations are used in liver disorders in ethno medicinal practices as well as traditional system of medicine in India. According to WHO about 18,000 people die every year due to Diabetic disease. Glycemiea diseases remain one of the serious health problems is the absence of variable liver protective drugs. The common ailments of liver are cirrhosis, cholestasis, hepatitis, portal hypertension, hepatic encephalopathy, hepatic failure and certain tumours like hepatoma. Various types of treatment modalities are available to treat Diabetic diseases. In allopathic medical practices, herbs play role in the management of various liver disorders. Since however, we do not have satisfactory remedy for disorders of sugar, the search for finding out effective diabetic drugs continues. Many unknown and lesser known plants are used is folk and tribal medical practices in India. The medicinal values of these plants are not known to the scientific world. The present work deals with Diabetic activity of ethanolic extract of leaf of Orthosiphon thymiflorens in Diabetic induced in rats. 11. Literature review of diabetes mellitus Glycaemia[14] In humans, blood glucose is tightly regulated by homeostatic mechanisms and maintained with in a narrow range. A balance is preserved between the entry of glucose into circulation www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2681 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences from the liver, supplemented by intestinal absorption after meals, and glucose uptake by peripheral tissues, particularly skeletal muscle. Hormonal influence on glycaemia The endocrine pancreas consists of about 1 million microscopic clusters of cells, the islets of langerhans. The islets in the adult human measure 100 to 200µm and consist of four major and two minor cell types. The four main types are: β-cells - secrete insulin α-cells- secrete glucagon δ-cells- secrete somatostatin Pancreatic polypeptide cells (ppcells) The two rare cells are D1 cells and enterochromaffin cells. Insulin Normal glucose homeostasis is tightly regulated by three inter related process: 1. Glucose production in the liver 2. Glucose uptake and utilization by the peripheral tissues, chiefly skeletal muscle 3. Ctions of insulin and counter-regulatory hormones, including glucagons. A rise in blood glucose levels results in glucose uptake in pancreatic β-cells, facilitated by an insulin-independent, glucose- transporting protein, GLUT-2.The resultant increase in intracellular ca2+ stimulates secretion of insulin, presumably from stored hormone with in the β-cell granules. Insulin is one of the most potent anabolic hormone known and its principle metabolic function is to increase the rate of glucose transport into certain cells in the body. In striated muscle cells (including myocardial cells), glucose is stored as glycogen or oxidized to generate ATP. In adipose tissue glucose is stored as lipid by insulin promoting lipid synthesis, and inhibiting lipid degradation. Similarly insulin promotes amino acid uptake and protein synthesis and inhibits protein degradation. Insulin cause most of the glucose absorbed after meal to be stored Somatostatin immediately in the liver in the form of glycogen. Then between meals, when food is not available and the blood glucose concentration begins to fall, insulin secretion decreases rapidly and the liver www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2682 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences glycogen is split back into the glucose, which is released back into the blood to keep the glucose concentration falling from too low. Glucagon Glucagon, a hormone secreted by the α-cells of the islets of langerhans when the blood glucose concentration falls, has several functions that are diametrically opposed to those of insulin. Most important of these functions is to increase the blood glucose concentration, an effect that is exactly opposite that of insulin. The major effect of glucagon on glucose metabolism is (1) break down of liver glycogen (glycogenolysis) and (2) increased gluconeogenesis in the liver. Both of these effects greatly enhance the availability of glucose to the other organs of body. Paradoxically, glucagon stimulates secretion of insulin from β- cells in man and it has been proposed that it is involved in the secretion of insulin after ingestion of glucose. Somatostatin The δ cells of islets of langerhans secrete the hormone somatostatin by the factors like increased blood glucose, increased amino acids, increased fatty acids and increased concentration of several of gastrointestinal hormones, released from upper gastrointestinal tract in response to food intake. It acts locally within the islets of langerhans themselves to depress the secretion of both insulin and glucagon. It decreases the motility of stomach, duodenum and gallbladder by decreasing both secretion and absorption in the gastrointestinal tract. Blood glucose regulation Glucose is the only nutrient that normally can be used by the brain, retina and germinal epithelium of the gonads in sufficient quantities to supply them optimally with their required energy. Therefore, it is important to maintain the blood glucose concentration at a sufficient high level even though most tissues shift to utilization of fat and proteins for energy in the absence of glucose. The blood glucose concentration not rise too high because of the reasons like cellular dehydration, glycosuria followed by diuresis by the kidney and vascular injury associated with uncontrolled diabetes mellitus. Normoglycaemic levels 80-90mg/100ml --- Fasting person 120-140mg/100ml --- During 1st hour after meals www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2683 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Hyperglycaemic levels >110mg/100ml --- Fasting glucose >200mg/100ml --- Random glucose Diabetes mellitus Diabetes mellitus is a chronic metabolic disorder characterized by deregulation in carbohydrate, protein and fat metabolisms caused by the complete or relative insufficiency of insulin secretion or insulin action. It is regarded as a main cause of serious maladies in the 21st century affecting people across the globe, irrespective of sex, age and socioeconomic status.[15] Lifestyle patterns in the industrialized societies comprise an increasing availability and ingestion of high calorie food in the prevalence of a sedentary lifestyle. These factors are emerging as the fundamental causes of this fast spread ―epidemic‖. The number of people with diabetes anticipates rising from current estimate of 150-220 million in 2010 and 300 million in 2025.[16] A survey depicts that 4% of the adults in India suffer from diabetes in the year 2000 and it is expected to increase to 6% by the year 2025.[17] Hyperglycaemia occurs because of uncontrolled hepatic glucose output and reduced uptake of glucose by skeletal muscle with reduced glycogen synthesis. When the renal threshold for glucose reabsorption is exceeded, glucose splits over in the urine (glycosuria) and causes an osmotic diuresis (Polyuria) which in turn results in dehydration, thirst and increased drinking (Polydipsia).[18] Figure 1: Pathological basis of diabetes mellitus. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2684 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Etiological classification of diabetes mellitus[19] 1. Type І diabetes mellitus (IDDM) Type І A -- Immune –mediated Type І B – Idiopathic 2. Type ІІ diabetes mellitus (NIDDM) 3. Genetic defects of β-cell function Maturity-onset diabetes of the young (MODY)caused by mutations MODY1-Hepatocyte nuclear factor 4α [HNF-4 α] gene mutations MODY2-Glucokinase gene mutations. MODY3- Hepatocyte nuclear factor1α [HNF-1α] gene mutations MODY4-Insulin promoter factor [IPF-1] gene mutations MODY5-Hepatocyte nuclear factor 1β [HNF-1β] gene mutations MODY6-Neurogenic differentiation factor 1[Neuro D1] gene mutations MODYx –Unidentified gene mutation(s) Mitochondrial DNA mutations 4. Genetic defects in insulin processing (or) insulin action Defects in proinsulin conversions Insulin gene mutations Insulin receptor mutations 5. Exocrine pancreatic defects Chronic pancreatitis Pancreatectomy Neoplasia Cystic fibrosis Fibrocalculous pancreatectomy 6. Endrochrinopathies Acromegaly Cushing syndrome Hyperthyroidism Pheochromocytoma www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2685 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Glucagonoma 7. Infections Cytomegalovirus Coxsakie virus B 8. Drugs Thyroid hormone Glucocorticoid α-interferon Protease inhibitors β-adrenergic receptors Thiazides Nicotinic acid Phenytoin 9. Genetic syndrome associated with diabetes mellitus Down syndrome Klein felter syndrome Turner syndrome Prader-willi syndrome 10. Gestational diabetes mellitus Type І diabetes mellitus Clinical - Onset: <20 years - Normal weight - Markedly decreased blood insulin - Anti islet cell antibodies - Ketoacidosis common Genetics - 30-70% concordance in twins - Linkage to MHC class II HLA genes www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2686 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Pathogenesis - Autoimmune destruction of β-cells mediated by T cells and humoral mediators (TNF, IL- 1, NO) - Absolute insulin deficiency Islets cells - Insulitis early - Marked atrophy and fibrosis - β-cell depletion Type II diabetes mellitus Clinical - Onset: >30 years - Obese - Increased blood insulin (early); normal to moderate decreased insulin (late ) - No anti-islet cell antibodies - Ketoacidosis rare; nonketotic hyperosmolar coma Genetics - 50-90% concordance in twins - No HLA linkage - Linkage to candidate diabetogenic genes (PPARγ, calpain10)b - Insulin resistance in skeletal muscle, adipose tissue and liver - β-cell dysfunction and relative insulin deficiency Islets cells - No insulitis - Focal insulitis and amyloid deposition - Mild β-cell deposition Monogenic forms of diabetes mellitus Monogenic causes of diabetes result from either a primary defect in β-cell function or a defect in insulin /insulin receptor signaling. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2687 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Maturity onset of diabetes of the young (MODY) It is a primary defect in β-cell function that occurs with out β-cell loss affecting either β-cell mass and /or insulin production. It is the outcome of a heterogeneous group of six distinct genetic defects. (MODY1, MODY2, MODY3, MODY4, MODY5 and MODY6). Mitochondrial diabetes Mitochondrial diabetes is caused by a primary defect in β-cell function associating with point mutations in a mitochondrial t RNA gene impairing mitochondrial ATP synthesis that leads to decreased insulin secretion. Diabetes associated with insulin gene or insulin receptor mutations Mutations that affect insulin processing from its precursor (proinsulin) or those that affect insulin structure and its receptor are a rare cause of diabetes. Insulin receptor mutations that affect receptor synthesis, insulin binding or receptor tyrosine kinase activity can, in rare case result in mild to severe insulin resistance and type II diabetes. Neither insulin gene nor insulin receptor mutations contribute significantly to the incidence of type II diabetes. Gestational diabetes Gestational diabetes typically is diagnosed during the half of pregnancy and occurs when the β-cell reserve is unable to counter balance the insulin resistance caused by placental hormones. Although gestational diabetes mellitus usually is asymptomatic, the consequences may be substantial. Fetal complications include stillbirth, macrosoma, increased risk of birth trauma, and neonatal hyperbilirubinemia and /or hypoglycaemia. Insulin resistance[20] Insulin resistance is defined as resistance to the effects of insulin on glucose uptake, metabolism or storage. Insulin resistance is a characteristic feature of most patients with type II diabetes and is an almost universal finding in diabetes individuals who are obese. Insulin resistance leads to decreased uptake of glucose in muscle and adipose tissues and an inability of the hormone to suppress hepatic gluconeogenisis. It may be acute or chronic. Acute insulin resistance is associated with surgical or other trauma, emotional disturbance or infection. Chronic insulin resistance may be due to genetic, insulin-receptor abnormalities, insulin antibodies and associated endocrine disorders like acromegaly, adrenal hypercorticism or pheochromocytoma. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2688 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Long term complications of diabetes Micro vascular/neuropathic complications Retinopathy - Cataract - Impaired vision Nephropathy - Renal failure Peripheral neuropathy - Sensory loss - Motor weakness Autonomic neuropathy - Postural hypotension - Gastrointestinal problems Foot disease - Ulceration - Arthropathy Macrovascular complications Coronary circulation - Myocardial ischemia/infarction Central circulation - Transient ischaemic attack - Stroke Peripheral circulation - Claudication - Ischaemia Experimental diabetes[21,22] Studies in animal models of diabetes have contributed greatly to the understanding of the etiology and pathogenesis of the disease. The establishment of co-relation between the disease process in the animal and the human beings is of greater significance. Research work on diabetes involves many advantages of using animals as experimental models involving various aspects of the disease etiology, its manufactured genetics, pathogenesis, complications etc., which can be understood efficiently. However the major limitations in www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2689 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences using animal models is that a perfect correlation between the disease in animal models and that in human beings could not be established as on animal model has similar characteristics and exact pathological mechanism as that of the human disease. Induction of experimental diabetes in experimental animals can be carried out in various ways by using Chemical diabetogens Surgically by partial pancreatectomy Viral induction Genetic manipulation by selective inbreeding. The most commonly used diabetogens for inducing both IDDM as well as NIDDM are listed out in Table 1. Table 1 1. Diabetogen Dose Animal 1 Alloxan 65-150mg/kg Rat 2 Streptozotocin 40- 60mg/kg Rat 3 Cyclosporine A 40mg/kg Rat 4 Lithium salts 4mEq/kg Rat 5 Dehydro ascorbic acid 650mg/kg three days Rat 6 Dehydro isoascorbic acid 1.5mg/kg Rat 7 Methyl alloxan 53mg/kg Rat 8 Ethyl alloxan 50-130 mg/kg Rat 9 Oxine & Dithizone 50mg/kg Rabbit 10 Sodium diethyl dithiocarbonate 0.5-1gm/kg Rabbit 11 Potassium xanthate 1gm/kg Rabbit 12 Uric acid 1gm/kg Rabbit Alloxan as a diabetogenic agent Introduction The compound was discovered by Justus von Liebig and Friedrich Wohler following the discovery of urea in 1828 and is one of the oldest named organic compounds that exist. Alloxan was originally isolated in 1818 by Brugnatelli and was named in 1838 by Wohler and Liebig. The name "Alloxan" emerged from an amalgamation of the words oipp" and "Oxalsäure" (oxalic acid). www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2690 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Chemistry O O HN O N O H The name is derived from allantoin, a product of uric acid excreted by the fetus into the allantois and oxaluric acid derived from oxalic acid and urea, found in urine. IUPAC name - 1,3 - Diazinane - 2,4,5,6 – tetrone Other names - Mesoxalylurea - 5 –Oxobarbituric acid Chemical properties Alloxan (2,4,5,6-tetraoxypyrimidine ; 2,4,5,6-pyrimidinetetrone) is - An oxygenated pyrimidine derivative. It is also a barbituric acid derivative (5- ketobarbituric acid). - Present as alloxan hydrate in aqueous solution. - A beta cell toxic glucose analogue with a molecular shape similar to that of glucose. - A very hydrophilic compound. - A week acid. - Chemically instable in bffer solutions with a half-life of 1.5 min at pH 7.4 and 370 C decomposing to alloxanic acid. - Stable at acid pH (0.01 M HCl). - A toxic thiol reagent. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2691 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences O O HO O NH HN + O N O O N O H H alloxan alloxantin O O O HN NH O N O O N O H H dialuric acid Figure 2 - It is reduced to dialuric acid in the presence of GSH and other thiols. - During redox cycling between alloxan and dialuric acid, new alloxan is formed continuously, so that toxic ROS species can be generated intracellularly over a long time period (>1 h). - During each redox cycle a small amount of ―compound 305‖, an alloxan –GSH adduct of unknown structure with characterstics absorbance at 305 nm that is not toxic, is formed. - A protoxin which generates in its xenobiotic metabolisam toxic ROS species superoxide .- radicals (O2 ), hydrogen peroxide (H2O2) and in the presence of an iron catalyst hydroxyl radical (OH),when it redox cycles with dialuric acid. Biological effects Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species. This causes an insulin-dependent diabetes mellitus (called "Alloxan Diabetes") in these animals, with characteristics similar to type 1 diabetes in humans. Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2692 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences through uptake via the GLUT2 glucose transporter. Alloxan, in the presence of intracellular thiols, generates reactive oxygen species (ROS) in a cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is initiated by free radicals formed in this redox reaction. One study suggests that alloxan does not cause diabetes in humans. Others found a significant difference in alloxan plasma levels in children with and without diabetes Type 1. Alloxan when injected into an experimental animal, induce a multiphasic blood glucose response, which is accompanied by corresponding inverse changes in the plasma insulin concentration as well as sequential ultrastuctural beta cell changes finally leading to necrotic cell death. A 1st transient hypoglycaemic phase lasting maximally 30 min starts within the first minutes after alloxan injection. This short-lived hypoglycaemic response is the result of a transient stimulation of insulin secretion, as documented by an increase of the plasma insulin concentration. The underlying mechanism of this transient hyperinsulinaemia is a temporary increase of ATP availability due to inhibition of glucose phosphorylation through glucokinase inhibition.This initial transient hypoglycaemic phase is not observed in the case of a streptozotocin injection because, at variance from alloxan, streptozotocin does not inhibit glucokinase. During these first five minutes after toxin exposure the beta cells show no morphological signs of damage. Upto one hour after exposure to both alloxan and streptozotocin, at a time, when normoglycaemia and normoinsulinaemia are prevailing, morphological beta cell changes are still minimal. Discrete changes in the form of a pale cytoplasm and beginning intracellular microvacuolisation, dilatation of the cisternae of the rough endoplasmic reticulum and mildly swollen mitochondria become visible after 20-30 min, apparent in particular on the ultrastructural level. At this stage the secretory granules are still normal in number and ultrastructure. These early signs can be considered potentially reversible intracellular lesions. This 2nd phase of the blood glucose response starts with a rise of the blood glucose concentration one hour after administration of the alloxan, while at the same time the plasma insulin concentration decreases. This is the first hyperglycaemic phase after the first hours and is accompained by decreased plasma insulin concentrations. These changes result from an inhibition of insulin secretion from the pancreatic beta cells, which the alloxan induce due to its beta cell toxicity. Morphologically this phase is characterised in the beta cells by www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2693 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences intracellular area, a reduction of insulin content and of the number of secretory granules as well as swollen mitochondria with a loss of cristae of the inner membranes. The observations are consistent with a defective mitochondrial energy production due to toxic action of the diabetogens and an inhibition of pre-proinsulin biosynthesis as well as processing,packaging and storage of insulin in the secretory granules. This is the underlying cause for the inhibition of insulin secretion and the resultant hyperglycaemia and hypo insulinaemia during this phase. The 3rd phase of the blood glucose response is again a hypoglycaemic phase typically 4-8 hours. It causes convulsions and may even be fatal without glucose administration , in particular when liver glycogen stores are depleted through starvation. This severe transitional hypoglycaemia is the result of the alloxan induced secretory granule and cell membrane rupture. These changes comprise not only ruptures of the plasma membrane but also of other subcellular organells, including the Golgi complex. The outer and inner membranes of the mitochondria also lose their structural integrity in this phase. Expression of functinally essential proteins such as GLUT2 glucose transporter and glucokinase is lost and insulin protein is undetectable. The beta cell nuclei are shrunken with condensed chromatin as signs of piknosis. Nuclei show no TUNEL positive staining. These changes are irreversible and highly characteristic for a necrotic type of beta cell death. The 4th phase of the blood glucose response is the final permanent diabetic hyperglycaemic phase. Morphological and ultra structural analyses document a complete degranulation and loss of the integrity of the beta cells within 12-24 to 48 hours after administration of the alloxan. Non-beta cells such as alpha cells and other endocrine and non-endocrine islet cell types as well as the extra pancreatic parenchyma remain intact. Proving the beta cell selective character of the toxic action of alloxan. Cell debris of the dying beta cells is removed by scavenger macrophages, which are not activated. After destruction of the beta cells, when survival through insulin supplementation is secured. So called end stage islets reside in the pancreas, exclusively composed of non-beta cells. All morphological features of the beta cell destruction is characteristic for a necrotic cell death. This is clearly at variance from the situation of auto-immune type 1 diabetes mellitus, www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2694 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences both in humans as well as in mouse and rat models of the disease, where beta cell demise is the result of apoptotic cell death without leakage of insulin from ruptured secretary granules. Mechanism of action The diabetogenic agent alloxan has two distinct pathological effects interfering with the physiological function of the pancreatic beta cells. It selectively inhibits glucose induced insulin secretion through its ability to specifically inhibit the glucokinase, the glucose sensor of the beta cell, and it causes a state of insulin- dependent diabetes mellitus through its ability to induce a selective necrosis of the beta cells. These two effects of alloxan can be assigned to specific chemical properties of alloxan. The common denominator of these effects is selective cellular uptake and accumulation of alloxan by the beta cell. Biological effect of alloxan due to hydrophilicity, chemical instability and glucose similarity of the alloxan molecule: Selective uptake and accumulation by the beta cell Alloxan is a hydrophilic and very instable chemical compound and the alloxan molecule has a molecularshape, which is very similar to that of glucose. Due to their hydrophilic characeter both alloxan and glucose do not penetrate the lipid bilayer of the plasma membrane. However, the molecular shape of alloxan is so similar to that of glucose, that the GLUT2 glucose transporter iin the beta cell plasma membrane accepts this molecule as a glucose analogue (glucomimetic) and transports it into the cytosol. Alloxan does not inhibit the function of the transporter within the biological lifetime of the chemical compound alloxan, so that the alloxan molecule can enter the beta cells through this protein pore unrestricted, where it is accumulated selectively and executes its biological effects. Biological effects of alloxan due to redox cycling and generation of toxic reactive oxygen species( ROX): Beta cell toxicity and diabetogenicity Alloxan diabetes is a form of insulin-dependent diabetes mellitus caused by a direct toxic effect upon the endocrine pancreas; occlusion of the arterial blood supply to the pancreas prevents access of the injected alloxan to the beta cells and thus diabetes. Diabetes is the result of the selective pancreatic beta cell toxicity of this compound which induces a necrosis of the beta cells. In order to destroy insulin-producing cells, alloxan must enter the cell. Due to its hydrophilicity it does not permeate the lipid barrier of the plasma membrane. However, due to its similarity with the glucose molecule, it can enter the cell via the low affinity GLUT2 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2695 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences glucose transporter in the plasma membrane. This has been proven by the fact that alloxan is not toxic to insulin-producing cells which do not express the GLUT2 glucose transporter. Alloxan has no immediate and direct inhibitory effect upon glucose transport. Since the half-life of alloxan is short, it must be taken up and accumulated quickly in the beta cell. After a few minutes, in aqueous solution, alloxan has been converted into non- diabetogenic alloxanic acid through spontaneous decomposition. Thus, alloxan is ineffective, when blood flow to the pancreas has been interrupted for a few minutes after alloxan injection. N-substituted alloxan derivatives with a long carbon side chain such as butylalloxan differ chemically in one respect from alloxan.In contrast to the hydrophilic all0xan, they are lipophilic. Butylalloxan, like alloxan, is thiol reactive and generates ROS and therefore can act in a similar manner. In particular beta cells are damaged preferentially, when isolated pancreatic islets are exposed to lipophilic alloxan derivatives. But as derivatives such as butylalloxan are lipophilic they can penetrate a plasma membrane without GLUT2 glucose transporter expression. Thus, these derivatives can enter all types and are systemically toxic rather then diabetogenic. Nephrotoxicity is a dominating feature of the toxicity of lipophilic derivatives after systemic administration. This nephrotoxiity, which develops in rats after injection, is so severe that it causes fatal renal failure in the animals before diabetes can develop. Though at the time of these early studies not known, the particular susceptibility of a preferential accumulation of these lipophilic toxins in the tubular cells of the kidney, which, like the pancreatic beta cells, express the GLUT2 glucose tranporter. Thus, experiments with lipopilic alloxan derivatives have added much experimental support to the concept that the hydrophilic character of the alloxan molecule is the beta cells and thus for its diabetogenicity. Alloxan can generates ―reactive oxygen species ― (ROS) in a cyclic reaction between this substance and its reduction product ,dialuric acid. The beta cell toxic action of alloxan (A) is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid (AH2) . generates superoxide radicals (O2 ) (Reaction 3-4) and hydrogen peroxide(H2O2)(Reactions 3- 4)and , in the Fenton reaction(Reaction 5) ,in the presence of a suitable metal catalyst(typical iron) (Reaction 4), hydroxyl radicals (.OH) (Reaction 5-7). The autoxidation of dialuric acid involves the intermediate formation of the alloxan radical (.AH) (Reaction 1-4) . .- + AH2 + O2 AH + O2 + H (1) . . AH + O2 A + O2 +H (2) www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2696 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences .- + . AH2+O2 +H AH + H2O2 (3) . .- + AH + O2 +H A+H2O2 (4) - . H2O2+e HO +HO (5) 2 3 . - Fe +H2O2 Fe +OH +OH (6) .- metal catalyst . - Net: O2 +H2O2 O2+OH + OH (7) Reduction of alloxan (A) to dialuric acid (AH2) in the cell requires the precence of a suitable thiol, typically the tripeptide glutathione (GSH) to generate the redox cycling partener (dialuric acid), while glutathione(GSSG) is oxidized (Reaction 8). A + 2GSH AH2 + GSSG (8) The reaction takes place in two steps with the alloxan radical (.AH) as an intermediate product (Reaction 9-10). A + GSH AH. + GS. (9) AH.+GSH AH+ GS. (10) Other intracellular thiols present at lower concentrations in the cell, such as monothiol cysteine and other thiols and dithiols as well as ascorbic acid, are also suitable reducing agents and thus may contribute, even though to a lesser extent, to alloxan reduction. Reaction with thiols and thus generation of ROS is also possible with proteins such as enzymes and albumin. During each redox cycle a small amount of ―compound 305‖, an alloxan-GSH adduct, is formed through reaction between alloxan and GSH. While the intracellular concentration of ―compound 305‖ increases in a time dependent manner, the amount of reduced GSH available in the cell for redox cycling diminishes gradually and thus fosters a lower pro-oxidative ratio between alloxan and GSH, rather than a higher anti- oxidative ratio. Thiols, cysteine as well as GSH, hav been reported long ago already to protect rats against development of alloxan diabetes, when injected together with alloxan. This, at a first glance, paradoxical observation can now be explained in the light of the understanding of the extracellular space are significantily induced extrecelluiarly so that less is available for accumulation in the beta cells, there by ameliorating the beta cell toxic and diabetogenic action of alloxan. Normally the capacity for reduction of alloxan, redox cycling and generation of ROS in the circulation is not sufficient to prevent the alloxan molecule to reach and enter the pancreatic beta cell. On the other hand through fostering of redox cycling in the organism the general systemic toxicity of alloxan is increased. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2697 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences .— The major oxidation pathway of dialuric acid, an o2 dependent chian reaction, is inhibited by superoxide dismutase (SOD) (Reaction 11). In the presence of SOD, an autocatalytic process involving the interaction between dialuric acid and alloxan becomes important, while iin the presence of a transition metal, a third oxidation mechanism, dependent upon H2O2,has been identified. This later step is inhibited by the hydrogen peroxide inactivating enzyme catalase. The other hydrogen peroxide inactivating enzyme, glutathione peroxidase(GPX), can principally act in a similar manner. But this enzyme requires GSH, which is oxidized in this reaction to GSSG (reaction 13). + .- 2H + 2O2 SOD H2O2 + O2 2H2O2 catalase O2 + 2H2O 2GSH + H2O2 2H2O + GSSG The alloxan molecule itself is not cytotoxic. When kept in the oxidized form and when reduction and/ redox cycling are prevented, alloxan does not generate ROS. Without ROS generation alloxan is not toxic to insulin-producing cells. The reduction product dialuric acid itself is also not toxic, when kept in the reduced form and when oxidation and redox cycling and/or generation of ROS is prevented. SOD and catalase prevent dialuric acid toxicity as documented by the protection against death of insulin- producing cells. At variance from alloxan, dialuric autooxidises spontaneously in the presence of O2, thus generating cytotoxic ROS in the absence of a thiol,even when not taken up into the cell. In contrast, alloxan, when restricted to the extra-GSH. Thus it is beta cell lesions as alloxan. Thiols in the plasma membrane, with which alloxan could interact and in the consequence could be reduced and generate ROS in a redox cycle, are apparently not present or not accessible to an extent, which would allow generation of ROS sufficient to damage the cells. Thus, a former hypothesis that alloxan might be cytotoxic due to interaction with thiol groups in the beta cell membrane is not valid. The antioxidative enzyme SOD greatly attenuates the toxicity of both alloxan and dialuric acid to insulin-producing cells in the presence of GSH. The reason for this cytoprotective - effect is the ability of SOD to scavenge superoxide radicals, which are generated in the O2 dependent chain reaction between dialuric acid and alloxan. The resultant suppression of dialuric acid autooxidation prevents ROS generation and through this mechanism counteracts www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2698 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences the toxicity to insulin-producing cells. Increasing the concentrations of the toxins, however, can reinstall the toxic action of both compounds. This is due to the fact that an autocatalytic reaction between the oxidized and reduced pyrimidine comes into action, which also generates ROS, when the chain reaction is suppressed by SOD. The superoxide radical, however, is apparently not the species responsible for the cytotoxicity of alloxan and dialuric acid, because the hydrogen peroxide(H2O2) inactivating enzyme catalase provided significantly better protection against the toxicity to insulin-producing cells .- than the superoxide radical(O2 ) inactivating enzyme SOD, though catalase does not prevent redox cycling and therefore does not prevent superoxide radical formation. This proves .- . convincingly that not the superoxide radical (O2 ) but rsther the hydroxyl radical( OH) is the ultimate toxic ROS species, whose formation from hydrogen peroxide(H2O2) is prevented through destruction of hydrogen peroxide by catalase. Optimal protection against the cytotoxic action of alloxan and dialuric acid towards insulin- producing cells, however provided only by a combination of SOD plus catalase. Only this combination completely prevents redox cycling between alloxan and dialuric acid and thus the generation of all ROS species in this chain reaction, namely superoxide radicals(O.2), . hydrogen peroxide(H2O2) and hydroxyl radicals( OH), which are ultimately responsible for cell death. A multitude of metal and in particular specific iron chelators as well as hydroxyl scavengers have been tested for their protective action both in vitro and in vivo. But though metal chelators such as EDTA, diethylenetriamine pentaacetic acid, desferrioxamine and phenanthroline and ROS scavengers such as the alcohols butanol and dimethylsulfoxide and the urea derivatives dimethylurea and thiourea have been shown in many experimental situations to provide protection against alloxan toxicity, the results obtained have never been entirely unequivocal. Due to the extremely short half-life and the extraordinary chemical reaactivety of the hydroxyl radical, it is not surprising that a complete protection against alloxan toxicity cannot be achieved, because hydroxyl radicals interact with biological targets before they can be inactivated through hydroxyl scavengers. In the same way, it is not surprising that has been extremely difficult to efficiently suppress metal catalysed hydroxyl radical formation through metal chelators, since it is crucial that metal chelation is achieved before hydroxyl racidal formations is initiated. Due to the chemical properties of the chelators and scavengers, this cannot always be achieved completely. Unless experimental conditions www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2699 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences are optimized. These conclusion is supported by the observation that the toxicity of alloxan and dialuric acid to insulin-producing cells in vitro is suppressed through the iron chelator desferrioxamine, which prevents the generation of very toxic hydroxyl radicals in the iron catalyzed fenton reation. Overall, the results with radical scavengers and metal chelators support the contention, that the hydroxyl radical is the ROS species ultimately responsible for alloxan-induced cell dealth. The intracellular GSH concentrations are in the same millimolar concentrations range in insulin-prodrucing cells as in liver cells. As both cell types express the GLUT2 glucose transporter a difference in the intracellular GSH concentration cannot explain tha mush greater susceptibility of insulin producing cells than of liver cells to the toxicity of alloxan in vivo. However, liver cells are much better endowed with the hydrogen peroxide inactivating enzyme catalase than insulin producing cells. When the low intracellular levels of calatase protein expression are rised in insulin producing cells through overexpression, this cells are protected equally well. This proves convincingly that the low hydrogen peroxide inactivating enzyme capacity is crucially responsible for the exquisite susceptilbility of insulin producing beta-cells towards alloxan toxicity. Thus the mechanism underlying the cytotoxin action of alloxan to insulin producing cells is due to reduction by interaction with intracellular thiols such as GSH and the resultant formation of cytotoxic ROS in a cyclic reaction between alloxan and its reduction product, dialuric acid, and hydrogen peroxide and ultimately, from the latter, the hydroxyl radical. Alloxan diabetes, induced in Laboratory animals through injection of this diabetogenic compound, is therefore the result of selective uptake of alloxan via the low affinity GLUT2 glucose transporter into a pancreatic beta cell, which due to its weak expression of hydrogen peroxide inactivating enzymes is badly protected against hydroxyl radical mediated cytotoxicity. Not surprisingly, therefore, effective prevention of redox cycling and generation of ROS or efficient inactivation of ROS can prevent beta cell death and counteract the development of alloxan diabetes in vivo. Thus, it can be concluded that the pancreatic beta cell toxicity and the resultant diabetogenicity of alloxan is due to the redox cycling and the generation of toxic reactive oxygen species(ROS) in combination with the hydrophilicity and the glucose similarity of the molecular shape of alloxan. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2700 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Oxidative stress[23,24,25] Oxidative stress, an imbalance between the generation of reactive oxygen species and antioxidant defense capacity of the body, is closely associated with aging and a number disease including cancer, cardiovascular diseases, neurological disorders, neurodegenerative diseases, diabetes and diabetic complications. Several mechanisms may cause oxidative insult in diabetes, although their exact contributions are not entirely clear. Accumulating evidence points to many interrelated mechanisms that increase production of reactive oxygen and nitrogen species or decrease antioxidant protection in diabetic patients. Advanced glycation or glycosylation end products (AGEs), the products of glycation and oxidation (glycoxidation) increase with age and at an accelerated rate in diabetes mellitus. In vitro studies have suggested that glycation itself may result in production of superoxide. Free Radicals and Their generation sites Hyperphysological burden of free radicals causes imbalance in homeostatic phenomena between oxidants and antioxidants in the body. This phenomenon leads to oxidative stress that is being suggested as a root cause of various human diseases. In modern western medicine the balance between antioxidation and oxidation is believed to be a critical concept for maintaining a healthy biological system. Research in recent past have accumulated enormous evidence advocating enrichment of body systems with antioxidants to correct vitiated homeostasis and prevent the onset as well as treat the disease fostered due to free radicals and related oxidative stress. Free radicals are also formed in the cells during normal metabolic processes, following exposure to ionizing radiation or drugs or xenobiotics. As molecular oxygen is available readily to accept electrons, oxygen centered free radicals are formed as the primary and secondary metabolites of cellular free radical reactions .The various cellular sites are plasma membrane, mitochaondria, peroxisomes, endoplasmic reticulum and plasma membrane. The reactive oxygen species include free radicals like ˉ O2 (superoxide) OH˚ (hydroxyl) HOO˚(hydroperoxy) ROO˚(peroxy) RO˚(alkoxy) www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2701 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The reactive nitrogen species include NO˚(nitric oxide) ONOOˉ (peroxynitrite) Other toxic halogen free radicals produced in the body are ˉ CCL3 CLˉ Oxidants[26,27,28] Superoxide anions Super oxide anion is highly reactive and toxic to cell membranes. When oxygen molecule ˉ. takes up one electron, by the univalent reduction, it becomes superoxide anion O2 It can capture further electrons to form hydrogen peroxide, H2O2, which is toxic and injurious. ˉ Whenever superoxide anion, O2 , is formed in tissues, it will lead to the formation of other free radicals like hydroperoxy radical, hydroxyl free radical and hydrogen peroxide. Formation of superoxide anion in metabolic pathways: Cytosolic oxidations by autooxidizable FP dependent enzymes like xanthine oxidase and aldehyde dehydrogenase.oxidative deamination by L-amino acid oxidase superoxide anion may be formed when reduced flavins are reoxidised univalently by molecular O2. It is also formed during univalent oxidations with molecular oxygen in respiratory chain. It can be formed during methaemoglobin formation. It may also form during cytosolic hydroxylation of steroids, drugs, and xenobitics, by Cy P450 or Cy P448 system. It is also produced during phagocytosis, by NADPH oxidase system during respiratory burst when O2 consumption is increased. Hydrogen peroxide (H2O2) Hydrogen peroxide is the most stable ROS. It is the less reactive and the most readily detected. H2O2 may be generated directly by divalent reduction of O2 or indirectly by univalent reduction of O2.H2O2 is the primary product of the reduction of O2 by numerous oxidases, such as xanthine oxidase, uricase and α-hydroxy acid oxidase localizes in peroxisome. The H2O2 is decomposed to H2O and O.H2O2 like most peroxides, is very sensitive to decomposition by the species that react with it. The reaction is catalysed by redox-active metal complexes, of which catalase and peroxidase are the most effective www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2702 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences exponents. Experiments with antioxidants enzymes show that hydrogen peroxide rather than super oxide anion is the most essential species to induce cell injury. H2O2 has been known to cause DNA damage in the form of chromosomal aberrations. Hydroxyl radical (OH) Hydroxyl is highly reactive. It can react with practically any molecule present in cells and is short lived. This insufficient stability does not allow it to diffuse through the cells. Therefore it reacts with organic substrates at the sites or near the sites of its formation. The life span of OH radical at 370˚C is 10-9 seconds. The reactions of OH radical are thus site specific. OH radical is produced following the reaction of O2 and H2O2 in presence of metallic ions like ferrous and copper ions. They are very susceptible to OH radical attack and initiates LPO.OH radical is the most potent among ROS reacting with a wide Lipid range of macromolecules at a high rate constant. OH radical is known to induce conformational changes in DNA including strand breaks base modifications. Nitric oxide (NO) Nitric oxide is an inorganic free radical gas, containing odd number of electrons and can form covalent link with other molecules by sharing a pair of electrons .It is synthesized by a family of isoenzymes called nitric oxide synthase located in various tissues and play an active role in free radical mediated diseases. It regulates numerous physiological process, including neurotransmission, smooth muscle contractility, platelet reactivity and the cytotoxic activities of immune cells. Moreover, it may have a role in carcinogenesis by inducing DNA strand breaks. NO can stimulate O2, H2O2, OH induced LPO. Lipid peroxidation (LPO) Lipid peroxidation (LPO) can be defined as the oxidative deterioration of lipids that contain a large number of carbon-carbon double bonds. Toxic byproducts called ―second messengers‖ are formed due to LPO, as membrane lipids are susceptible to it. Since membranes form the basis of cellular organelles like mitochondria, endoplasmic reticulum, plasma membrane, peroxisomes, lysozomes etc, the damage caused by the LPO is highly detrimental to the functioning of the cell and its survival LPO, alters the biophysical properties of the cell membranes, decreases the membrane fluidity and decreases the electrical resistance .Cross linking also occurs which resists the mobility of the membrane proteins. Leakage of cytosolic enzymes may also occur on extensive peroxidative attack. An iron induced lipid peroxidation process is described as peroxidative sequence initiated by the attack of an unsaturated lipid by www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2703 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences any species that abstracts hydrogen atom from a methylene group which leaves an unpaired electron to the carbon atom. The resultant carbon radical is stabilized by molecular rearrangement to produce a conjugated diene. It readily reacts with an oxygen molecule forming peroxyl radical that abstracts hydrogen from he lipids which is further degraded in presence of iron and breaking into malondialdehyde and other such end products. Free radical effect on biomembranes Free radicals are highly reactive so can initiate chain reaction and brings about lipid peroxidation producing lipid peroxides and lipoxides. These radicals constitute a threat to the integrity of biomembranes which could be oxidized. The free hydroxyl radical is most reactive and can also be mutagenic an extraordinarily potent oxidant. These oxidants can oxidize –SH group containing membrane proteins in the cells and biomembranes to S-S group, methionine to its sulphoxide and membrane lipids, unsaturated FA to lipid peroxides and lipoxides. All these events will affect the optimum fluidity of the membrane causing membranopathy.O2ˉ and OH˚ can initiate chain reaction and bring about oxidation of polyunsaturated FA of membranes. Antioxidants Antioxidants are substances or compounds used to reduce lipid peroxidation both in humans and in nature. Antioxidants can be classified into two ways based on their mode of action and source of availability. 1. According to their mode of action: Preventive antioxidants These reduce the rate of chain initiation. Catalases Other peroxides that can react with ROOH Natural endogenous caeruloplasmin, Chelators of metal ions such as : . DTPA(diethylene tri amine penta acetate),and . EDTA(Ethylene diamine tetra acetate) Chain breaking antioxidants They interfere with chain propagation. Phenols or aromatic amines. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2704 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences In vivo the principle chain breaking antioxidants are: . Superoxide dimutase both cytosolic and mitochondrial . Vitamin E and Selenium-containing glutathione peroxidase . Urates Peroxidation is also catalyzed by in vivo by heme containing compounds and by lipooxygenases found in platelets and leucocytes. Synthetic enzyme as antioxidant Recently researchers have prepared a synthetic enzyme called ―synzyme‖ which works just like the body’s own scavengers like superoxide dismutase (SOD), to mop up ―free‖ radicals. The compound is more powerful than traditional antioxidants such as vitamin E or vitamin C. 2. Based on their source of availability Naturally occurring antioxidants Lipid soluble: . Vit E (tocopherol) . β-carotene: is an antioxidant at low PO2 . Lycopene Water soluble: . Vitamin C (Ascorbic acid) . Urates Antioxidants used in vitro: These are used to prevent lipid peroxidation in foods. Propyl galate (PG) Butylated hydroxyl anisole (BHA) Butylated hydroxyl toluene (BHT) Enzyme systems Superoxide dismutase (SOD) Superoxide dismutase (SOD) is a major antioxidant enzyme. SOD exists in different isoforms. Cu, Zn-SOD is mostly in the cytosol and dismutases superoxide to hydrogen peroxide. Extracellular (EC) SOD is found in the plasma and extracellular space. Mn-SOD is located in mitochondria. Decreased activity of cytoplasmic Cu, Zn-SOD and especially mitochondrial (Mn-) SOD in diabetic neutrophils wad found. As a result of this superoxide www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2705 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences levels were elevated in neutrophils of diabetic patients. Major reason for the decreased SOD activity is the glycosylation of Cu, Zn-SOD which has been shown to lead to enzyme inactivation both in vivo and in vitro. Also Cu, Zn-SOD cleavage and release of Cu++ in vitro resulted in transition metal catalyzed ROS formation. Catalase Catalase is a hydrogen peroxide decomposing enzyme mainly localized to peroxisomes or microperoxisomes. Catalase decreases the cross linking and AGE formation and prevents the formation of superoxide. It is an enzyme of heme protein and each enzyme sub unit also contains one molecule of NADPH, which helps to stabilize the enzyme .Dissociation of protein subunit results in loss of enzyme activity. Figure 3 Mechanisms for increased oxidative stress in diabetes mellitus ROS; reactive oxygen species, GSH; reduced glutathione, GSSG; oxidized glutathione, GRD; glutathione reductase, GPX; glutathione peroxidase, AR; aldose reductase. Glutathione dependent enzymes Tissue glutaothione plays a central role in antioxidant defense. Reduced glutathione detoxifies reactive oxygen species such as hydrogen peroxide and lipid peroxides directly or in a glutathione peroxide (GPX) catalyzed mechanism. Glutathione also regenerates the major www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2706 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences aqueous and lipid phase antioxidants, ascorbates and α-tocopherol. Glutathione reductase (GRD) catalyzes the NADPH dependent reduction of oxidized glutathione, serving to maintain intracellular glutathione stores and a favourable redox status. Glutathione -S- transferase (GST) catalyzes the reaction between the –SH group and potential alkylating agents, rendering them more water soluble and suitable for transport out of the cell. GST can also use peroxides as a substrate. The polyol pathway Hyperglycaemia induces the polyol pathway, reduction in induction of aldose reductase and production of sorbitol.Importance of polyol pathway may vary among tissues.In type II diabetes mellitus patients activation of polyol pathway appears to deplete erythrocyte NADPH and GSH. III. Literature review of plant Plants have been used in traditional medicine since ancient times for the treatment of various diseases of man and animals. However, still a large number of local herbs claimed to be useful in the treatment of many diseases including diabetes have not been screened. In addition, increased awareness to the unwanted effects of allopathic drugs has encouraged people to look alternative drugs 29. Traditional medicines and herbs would probably open new therapeutic venues for multifactorial disease, such as diabetes mellitus, since their complex complications often provide versatile bioactivity and varied mechanism of action. Indian traditional medicine is one of the richest medicinal systems among those available around the world. Long before the used of insulin, since the time of charaka and sushrutha (sixth century BC, 400 BC), indigenous medicines have been used for the treatment of diabetes mellitus. In accordance with the recommendations of the WHO expert committee on diabetes mellitus, an investigation of antihyperglycaemic agents of plant origin used in traditional medicines seems important. Many herbs and plant products have been shown to have antihyperglycaemic action. Extract of drugs from plant sources such as Allium sativum (garlic) Azadirachta indicia (neem) Vinca rosea (nayntara) Gymnea sylvestra (meshashringe) Trigonella foenum greaceum (fenugreek) Momordica charantia (bittergourd) www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2707 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Ficus bengalensis (banyan) Eugenis jamobolana (black berry) Ocimum santum Linn (tulsi) Eclipta Alba (karichalankanni) are some of the plants reported to possess antihyperglycaemic activity in experimental animals.[30] One such ethno botanically important plant, Orthosiphon thymiflorus(Roth), a plant drug of traditional systems of medicine in India i.e. Ayurveda and Siddha is used for the treatment of diabetes mellitus. An effort to pharmacologically evaluate the plant for its anti-diabetic and antioxidant property is done in the present study. Figure 4: Orthosiphon thymiflorus roth. Plant taxonomy[31,32,33,34] Kingdom: Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Asterids Order: Lamiales Family: Lamiaceae Genus: Orthosiphon Sanskrit synonyms: Pratanika Ayurvedic properties rasa: Tikta, Katu Guna: Lakhu. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2708 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Virya: Ushna Plant name in different languages Malayalam: Chilannippadam, Chilannippatam Distribution – Throughout south India, in dry open forests, in Talakona forest, in Chittoor District of Andhra Pradesh, India. Plant description: A perennial woody shrub grows up to 1 meter in height. Leaves simple, opposite, long petiolate, decussate, broadly ovate, acute, obtuse or subcordale at base, coarsely crenale or serrate, nearly glabrous, flowers in whorls in terminal racemes. Fruits 4, dry ellipsoid smoothnutlets. Medicinal properties Orthosiphon is used for treating the ailments of the kidney, since it has a mild diuretic effect. It is also claimed to have anti-allergenic, anti-hypertensive and anti-inflammatory properties, and is commonly used for kidney stones and nephritis. Orthosiphon is sometimes used to treat gout, diabetes, hypertension and rheumatism. It is reportedly effective for anti-fungal and anti-bacterial purposes. Useful part: Whole plant. Past work done on this plant The effect of aqueous extract of orthosiphon thymiflorus on isolated skeletal muscles. Antioxidant Potential of leaf extract of Orthosiphon thymiflorus (Roth.) In-vitro Cytotoxic Activity of Orthosiphon thymiflorus Roth.) Sleensen Leaf extract against Dalton Lymphoma Ascites Cell Line. IV. Scope and Plan of work Scope of work Management of diabetes mellitus is a global problem and successful treatment is very essential for preventing or at least delaying the onset of long-term complications of the disorder. Remedies to treat such chronic states are available in nature in the form of herbal medicines or drugs with very minimal adverse effects when compared to the available synthetic drugs. Such herbal drugs as therapeutic agents are a boon when compared to the severe adverse effects of the allopathic medical practice for diabetes, though the quest for a complete and permanent cure for the disease is being pursued relentlessly by eluding physicians and researchers.[35] These herbal remedies which exemplifies the process of www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2709 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences symbiosis still remains unexplored amidst the modern technical advances, which has created a tremendous scope for folk lore medicines. It is believed that the traditional medicines used for the treatment of diabetes mellitus attenuate the progression of complications of the disease.[36] scientific validation of such plants is necessary, because though their medicinal use dates back to 1000 years, a scientific literature that supports it’s therapeutic and pharmacological properties become necessary. The search for the effective herbal drugs for the treatment of diabetes based on ethnomedical clues still continues and in the long run has yielded us invaluable herbal remedies. To prove the ethno medical use of such folkloric traditional medicines, we have selected such ethno botanically important plant, Orthosiphon thymiflorus Roth a plant used in the traditional systems of medicine in India for the treatment of diabetes mellitus to pharmacologically evaluate the plant for its antidiabetic property along with it’s antioxidant potential in the present study. Plan of work The following studies were done on the ethanolic extract leaf of Orthosiphon thymiflorus Roth 1. Preparation of extracts. 2. Preliminary photochemical analysis 3. Acute oral toxicity studies. (OECD 423guidelines) 4. Effect on blood glucose levels on normoglycaemic and glucose fed hyperglycaemic rats. 5. Effect of sub-acute treatment on body weights in Alloxan induced -diabetic rats. 6. Effect of sub-acute treatment on blood glucose leveling Alloxan induced- diabetic rats. 7. Effect on serum biochemical parameters in Alloxan induced diabetic rats. Total protein in serum Total cholesterol in serum HDL-cholesterol in serum Triglycerides in serumLow density lipoprotein (LDL) Very low density lipoprotein (VLDL) Serum creatinine. 8. Histopathalogical studies of pancreas. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2710 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences V. MATERIALS AND METHODS A. Materials Collection and authentication of plant material The leaf of Kyllinga nemoralis was purchased from Mr. Madhusudhan. The plant material was identified and authenticated by registered botanist .A voucher specimen was submitted at college. Preparation of plant extract Extraction Extraction can be defined as the removal of soluble materials from an insoluble residue, either liquid or solid, by treatment with a liquid solvent. In the process of successive extraction the solvent from low polarity to high polarity is used and the apparatus used Soxhlet apparatus and thus process is known as Soxhlation. Steps involved in the extraction of crude drugs - Suitable size reduction of the crude drugs. - Penetration of the crude drug by the solvent. - Dissolution of the soluble matter within the cells. - The escape of the dissolve material through the cell walls and through the solvent, boundary Layer surrounding the particles of the crude drug. - Separation of the solution and removal of the exhausted drug. - Removal of solvent to obtain the extract Preparation of ethanolic extract of the leaf of Orthosiphon thymiflorus Roth (EEOT) - The collected plant materials were dried in shade for about 15 days, made in to coarse powder with the use of mixer grinder. Powdered plant material was extracted with and Ethanol in soxhlet apparatus for 24 hours. The extracts were collected and concentrated under reduced pressure to a semisolid mass. The extracts obtained dried in desicator. The dried extracts weighed and the percent yield was caliculated. Experimental animals Inbred adult wistar albino rats (150-280 g) of either sex were obtained from animal house of ------and the research work was carried out at same University. The animals were maintained in a well-ventilated room with 12:12 hour light/dark cycle in polypropylene cages. Standard pellet fed and tap water was provided ad libitum through out experimentation www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2711 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences period. Animals were acclimatized to laboratory conditions one week prior to initiation of experiments. Fasting refers to that the animals were deprived of food for 16 hours but were allowed to free access for water. Chemicals All the chemicals used in the study were of analytical grade. The following chemicals were used for the experimental study. Table 2: List of chemicals. Material Source 5-5’- Dithio-bis-2-nitrobenzoic acid Sigma chemical Co., USA (DTNB) Acetic acid S.d.fine chemicals Ltd, Mumbai. Alloxan S.d.fine chemicals Ltd,Mumbai Chloroform S.d.fine chemicals Ltd, Mumbai Med source Ozone Biochemicals Cholesterol kit Ltd,Faridabad. Med source Ozone Biochemicals Creatinine kit Ltd,Faridabad Ethylene diamine tetra acetic acid( Qualigens fine chemicals, Mumbai EDTA) FeCl3 reagent S.d.fine chemicals Ltd, Mumbai Glutamate dehydrogenase(enzyme) Sigma chemicals company, U.S.A solution from bovine liver (GIDH) Glucose Thermo electron LLS India Ltd. Glucose test strips Accuchek Ltd Hydrogen peroxide solution Qualigens fine chemicals, Mumbai n-butanol S.d.fine chemicals Ltd, Mumbai Nicotinamide adenine dinucleotide S.d.fine chemicals Ltd, Mumbai (NAD) Pyrogallol S.d.fine chemicals Ltd, Mumbai Potassium dihydrogen phosphate S.d.fine chemicals Ltd, Mumbai Perchloric acid Loba chemicals Ltd, Mumbai Reduced glutathione (GSH) Qualigens fine chemicals, Mumbai Sodium dodecyl sulphate (SDS) S.d.fine chemicals Ltd, Mumbai Sodium hydrogen carbonate S.d.fine chemicals Ltd, Mumbai Sodium acetate S.d.fine chemicals Ltd, Mumbai Sodium thiosulphate S.d.fine chemicals Ltd, Mumbai Thionyl chloride S.d.fine chemicals Ltd, Mumbai Thiobarbituric acid Rolex laboratory reagent, Mumbai Med source Ozone Biochemicals Ltd, Total protein kit Faridabad Ranbaxy laboratories Ltd. Chemical division, Trichloro acetic acid New Delhi Med source Ozone Biochemicals Ltd, Triglycerides kit Faridabad www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2712 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences B. Methods Part I Priliminary phytochemical analysis[37,38] The ethanolic extract of leaves were subjected to different chemical tests separately for the identification of various active constituents. The phytochemical constituents of plant of orthosiphon thymiflorus Roth results was recorded in table no: 3 Test for alkaloids Mayer’s test To the 1 ml of the extract, a drop or two drop of Mayer’s reagent was added by the side of test tube. A white or creamy precipitate indicates the test as positive. Wagner’s test To 1 ml of the extract, few drop of Wagner’s reagent was added. A reddish brown colour indicates the test as positive. Hager’s test To the 1 ml of the extract, few drop of Hager’s reagent was added. A prominent yellow colour indicates the test as positive. Dragendroff’s test To the 1 ml of extract, few drop of Dragendroff’s reagent was added. A prominent yellow colour indicates the test as positive. Test for carbohydrates Benedict’s test To the 5 ml of Benedict’s reagent, 1 ml of the extract solution was added and boiled for 2 minute and cooled. Formation of red precipitate shows the presence of carbohydrate. Molisch’s test To the 2 ml of extract, two drops of alcoholic solution of ά-nephthol was added and shaken well. 1 ml of concentrated sulphuric acid was added slowly along the side of the test tubes and allowed to stand. A violet ring indicates the presence of carbohydrates. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2713 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Fehling’s test To the 1 ml of extract, add equal quantity of Fehling solution A and B. A red precipitate indicates the presence of sugar. Barfoed’s test To the 2 ml of extract, 2 ml of Barfoed reagent was added and mixed well. It was heated for 1-2 minute in boiling water bath and cooled. Formation of red precipitate indicates the presence of sugar. Test for Protein and Amino acid Million’s test To the 2 ml of extract, few drops of Million reagent was added. A white precipitate indicates the presence of proteins. Ninhydrin test To the 2 ml of extract, two drops of Ninhydrin solution was added. A characteristic purple colour indicates the presence of amino acids, proteins and peptides. Biuret test To the 1 ml of extract, one or two drop of 1% copper sulphate solution was added and to this 1 ml of ethanol (95%) was added, which was followed by excess of potassium hydroxide pellets. The pink layer in ethanolic layer indicates the presence of proteins. Xanthoprotein test To the 1 ml of extract, add 1 ml of concentrated Nitric acid. A white precipitate was formed, boiled and cooled. Then 20% sodium hydroxide in ammonia was added. Orange colour indicates the presence of aromatic amino acid. Test for glycosides Legal’s test To the 2 ml of extract was dissolve in the solution of pyridine and to it sodium nitropruside was added, to make it alkaline. The formation of pink to red colour shows the presence of glycosides. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2714 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Baljet’s test To the 1 ml of extract, 1 ml of sodium picrate solution was added. The changing of colour from yellow to orange reveals the presence of glycosides. Borntrager’s test To the 1 ml of extract, few ml of sulphuric acid was added. It was boiled and filtered and extracted with chloroform. The chloroform layer was than treated with few ml of ammonia. The formation of red colour shows the presence of anthraquinone glycosides. Keller killani test The extract was dissolved in acetic acid containing the traces of ferric chloride and then it was transferred to a test tube containing sulphuric acid. At the junction, the formation of reddish brown colour, which gradually become blue, confirm presence of glycosides. Test for flavanoids Shinoda test To the 1 ml of extract, add magnesium turnings was added and 1-2 drops of concentrated hydrochloric acid was added drop wise. Formation of pink to crimson colour indicates the presence of flavanoids. Alkaline reagent test The aqueous solution of the extract was treated with 10% ammonium hydroxide solution. Yellow fluorescence indicates the presence of flavanoids. Test for Tannins and Phenolic compounds Ferric chloride test To the 1 ml of extract, add few drops of neutral 5% ferric chloride solution. Formation of dark greenish colour shows the presence of phenolic compounds. To the extract add potassium dichromate solution, formation of a precipitate shows the presence of tannins and phenolic compounds. Test for triterpenoids Two or three granules of tin metal were added in thionyl chloride solution in a test tube. Then 1 ml of extract solution was added into it. The formation of pink colour indicates the presence of triterpenoids. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2715 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Test for saponins The 1 ml of the extract was diluted with distilled water and the volume was made up to 20 ml. The suspension was shaken in a graduated cylinder for 15 minutes. 2 cm. layer foam indicates the presence of saponins. Test for fixed oil Spot test A small quantity was pressed between two filter papers. Oil stain on the paper indicates the presence of fixed oil. Saponification test A few drops of 0.5 N alcoholic potassium hydroxide solution was added to a small quantity of extract along with a drop of phenolphthalein. The mixture was heated on water bath for 2 hrs. Formation of the soap or partial neutralization of alkali indicates the presence of fixed oil. Test for steroids Libermann-Burchard test Dissolve the extract in 2 ml of chloroform in a dry test tube. Add 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid. The solution becomes red, then blue and finally bluish green, indicating the presence of steroids. Salkowski test Dissolve the extract in chloroform and add equal volume of concentrated sulphuric acid. Formation of bluish red to cherry red colour in chloroform layer and green fluorescence in the acid layer represented the steroid components in the tested extract. Part II Toxicological evaluation. Acute oral toxicity study The acute oral toxicity study was done according to OECD 423 guide lines (Acute toxicity class method). A single administration of a starting dose of 2000 mg/kg bw/p.o, of EEOT was administered to 3 male rats and observed for 14 days. There was no considerable change in body weight before and after treatment of the experiment and no signs of toxicity were observed. The results are shown in Table 4. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2716 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Experimental procedure[39] Wistar rats of male sex weighing 150-280g were used for the study. The starting dose level of EEOT was 200mg/kg body weight p.o. As most of crude extracts possess LD50 value more than 2000mg/kg p.o. Dose volume was administered 0.5ml/100mg body weight to over night fasted rats with 0.5%w/v SCMC. Food was with held for further 3-4 hours after administration of EEOT and observed for signs of toxicity. Body weight of rats before and after termination were noted and any changes in skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system and somato motor activity and behavior pattern were observed, and also signs of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma were noted. The onset of toxicity and signs of toxicity also noted. Flow chart for acute toxic class method (OECD guideline 423) starting dose of 2000 mg/kg body weight/po www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2717 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Part III Pharmacological studies Induction of diabetes mellitus in experimental animals[40,41] Adult inbred male wistar albino rats (32 numbers) of either sex were over night fasted and received a freshly prepared solution of alloxan, [S.d.fine chemicals Ltd], (150 mg/kg) in distilled water injected intraperitonially in a volume of 1 ml/kg .After injection the animals had free access to food and water and were given 5% glucose in their drinking water for the first 24 hours to counter any initial hypoglycemia. Normal rats (6 numbers) received 1ml distilled water as vehicle. The development of diabetes was confirmed after 48 hours of the alloxan injection. The animals with fasting blood glucose level more than 200 mg/dl were selected for the experimentation. Out of 32 animals subjected for diabetes induction, 6 animals died before grouping and two animals were omitted from the study, because of sub diabetic condition (118mg/dl) and (122mg/dl). Of the remaining 24 animals,4 groups of 6 animals were formed and used for the experimentation. In the present study, glibenclamide (0.4 mg/kg body weight) was used as the standard drug. Determination of the blood glucose levels Blood was collected from tip of the tail vein and fasting blood glucose level was measured using single touch glucometer (Accuchek Ltd) based on glucose oxidase method. Effect of EEOT on normoglycaemic and glucose fed-hyperglycaemic rats [NG-OGTT][40] A combined methodology is preferred for the activity assessment of extract in order to avoid wasting animals; there are some modifications incorporated in the time pattern for blood glucose level determination. After overnight fasting (16 h) the blood glucose level of rats were determined and then were given the test samples and standard. The animals were dived in to four groups of 6 rats in each. Group I - Animals received distilled water.. Group II - Animals received glibenclamide 0.4mg/kg b.w/p.o. Group III - Animals received EEOT 200mg/kg b.w/ p.o. Group IV - Animals received EEOT 400mg/kg b.w /p.o. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2718 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Test samples and standard were given immediately after the collection of initial blood samples. The blood glucose levels were determined in the following pattern: 30 and 60 min to access the effect of test samples on normoglycaemic animals. The rats were then loaded orally with 2g/kg glucose and the glucose concentrations were determined at 60, 90 and 210 min after glucose load. Effect of sub-acute treatment of EEOT on changes in body weight in Alloxan induced diabetic rats[42] The animals were dived in to 5 groups .Group I consists of normoglycaemic rats. The remaining 4 groups consisted of 6 Alloxan induced diabetic rats. Group I - Normal control animals received distilled water. Group II- Alloxan (150 mg/kg b.w) induced animals distilled water Group III -Alloxan (150 mg/kg b.w) induced animals received glibenclamide 0.4 mg/kg b.w/ p.o for 14 days. Group IV - Alloxan (150 mg/kg b.w) induced animals received EEOT 200 mg/kg b.w/ p.o for 14days. Group V - Alloxan (150 mg/kg b.w) induced animals received EEOT 400 mg/kg b.w p.o for 14 days. The above mentioned treatment schedule was followed for the respective group of animals for 14 days. Changes in the body weight was on 0th, 10th and 15th day of treatment. Effect of sub-acute treatment of EEOT on blood glucose level in Alloxan induced diabetic rats The animals were dived in to 5 groups .Group I consists of normoglycaemic rats. The remaining 4 groups consisted of 6 Alloxan induced diabetic rats. Group I - Normal control animals received distilled water. Group II - Alloxan (150 mg/kg b.w) induced animals received distilled water. Group III- Alloxan (150 mg/kg b.w) induced animals received glibenclamide 0.4 mg/kg b.w/ p.o for 14 days. Group IV - Alloxan (150 mg/kg b.w) induced animals received EEOT 200 mg/kg b.w /p.o for 14days. Group V - Alloxan (150 mg/kg b.w) induced animals received EEOT 400 mg/kg b.w/p.o for 14 days. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2719 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The above mentioned treatment schedule was followed for the respective group of animals for 14 days. Blood samples were collected from overnight fasted animals on 0th, 10th and 15th day to estimate blood glucose levels using glucometer. At the end of the study, all the animals were sacrificed under light ether anaesthesia. The rats were sacrificed by decapitation and blood was collected by bleeding of carotid artery and serum was separated to study the biochemical parameters. The relevant organs like liver and pancreas were removed dissected out and washed with ice-cold saline. The pancreatic tissues were preserved in 10% Formalin solution for histopathological studies. Biochemical studies Estimation of total protein Total protein level in serum was estimated by using protein test kit. Estimation of total cholesterol Total cholesterol was estimated in the serum by using test kit. Estimation of HDL-cholesterol HDL-cholesterol was estimated in the serum by using test kit. Estimation of triglycerides Triglyceride levels were estimated by using test kit. Estimation of LDL-cholesterol[43] LDL-cholesterol was calculated by using the formula LDL-cholesterol = total cholesterol − [HDL-C + (triglycerides/5) ] Estimation of VLDL- cholesterol[44] VLDL-cholesterol was caliculated by using the formula VLDL- cholesterol = (triglycerides /5 ) Estimation of creatinine Serum creatinine levels were estimated by using test kit. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2720 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences VI. RESULTS Preliminary phytochemical analysis of ethanolic extract of leaf of orthosiphon thymiflorus Roth The result of preliminary phytochemical analysis of ethanolic extract of leaf of orthosiphon thymiflorus Roth is shown in Table 3. Ethanolic extract showed the presence of flavonoids and steroids. Acute oral toxicity study The acute oral toxicity study was done according to OECD 423 guide lines (Acute toxicity class method). A single administration of a starting dose of 2000 mg/kg bw/p.o, of EEOT was administered to 3 male rats and observed for 14 days. There was no considerable change in body weight before and after treatment of the experiment and no signs of toxicity were observed. The results are shown in Table 4. Effect of EEOT on blood glucose levels in normoglycaemic and glucose induced hyperglycaemic rats. [NG-OGTT] The EEOT at a dose level 200mg/kg b.w/p.o did not exhibit significant hypoglycaemic effect in fasted normal rats after 30 minutes of administration and a high dose of 400mg/kg b.w/p.o reduced blood glucose in normal rats significantly after 60 min of drug administration (p<0.01). In the same group of rats which are loaded with glucose (2gm/kg b.w/p.o) after 60 min of drug administration a low dose of 200mg/kg bw reduced blood glucose level with less significance (p<0.05) but a high dose of 400mg/kg/b.w reduced blood glucose significantly (p<0.01) .The standard drug glibenclamide (0.4 mg/kg b.w/p.o) treatment showed significant reduction in blood glucose levels in both normal and glucose induced hyperglycaemic rats (p<0.01). Results are shown in Table-5 and Figure-5. Effect of sub acute treatment of EEOT on body weight changes in Alloxan induced diabetic rats The EEOT at oral dose level of 200mg/kg do not show significant improvement in the body weight of Alloxan induced diabetic rats on 10th day of the treatment and shows a slight significance in the body weight improvement on 15th day (p<0.05). An oral dose of 400mg/kg b.w shows significant improvement in the body weight of Alloxan induced diabetic rats on 10th day and 15th day of treatment (p<0.01). The standard drug glibenclamide (0.4 mg/kg b.w www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2721 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences /p.o) also produced significant improvement in body weight of Alloxan induced diabetic rats.(p<0.01). Results are shown in Table 6 and Figure 6. Effect of sub-acute treatment of EEOT on blood glucose level in Alloxan induced diabetic rats In the sub-acute study, Alloxan induced diabetic rats were treated with EEOT 200mg and 400 mg/kg b w.t /p.o for a duration of 14 days. Treatment with EEOT 200mg significantly (p<0.01) decreased the blood glucose level after 14th day onwards. Treatment with EEOT 500mg produced a significant (p<0.01) drop in blood glucose level after 10th day onwards. Treatment with glibenclamide 0.4 mg/kg produced a significant (p<0.01) decrease in blood glucose level after 10th onwards and thereafter. Results are shown in Table 7 and Figure 7. Biochemical estimations Serum total protein The diabetic control animals showed significant decrease in serum total protein level when compared to control animals. The serum total protein levels in EEOT (200mg and 400mg/kg b.wt) treated diabetic rats showed significant increase (p<0.05) and (p<0.01) respectively. Glibenclamide (0.4 mg/kg/p.o) showed significant (p<0.01) when compared to Alloxan induced diabetic rats. Results are shown in Table 8 and Figure 8. Serum total cholesterol The total cholesterol level significantly (p<0.01) increased in Alloxan induced diabetic rats when compared to control rats. Serum total cholesterol levels of diabetic animals treated with EEOT (200 and 400mg/kg b.w/p.o) showed significant (p<0.01) decrease in cholesterol level when compared to Alloxan induced diabetic animals. Results are shown in Table 9 and Figure 9. Serum HDL-cholesterol The serum HDL-cholesterol level was significantly (p<0.01) in Alloxan induced diabetic rats when compared to control rats. HDL-cholesterol level of diabetic rats treated with EEOT (200 and 400mg/kg b.wt/p.o)was significantly(p<0.05) and (p<0.01) increased respectively. However glibenclamide (0.4mg/kg b.w/p.o) treatment showed significant (p<0.01) increase HDL-cholesterol when compared to Alloxan induced diabetic animals. Result are shown in Table 10 and Figure 10. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2722 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Serum triglycerides The serum triglyceride level was significantly (p<0.01) increased in Alloxan induced diabetic rats when compared to control rats. Triglyceride level of diabetic rats treated with EEOT (200 and 400mg/kg b.wt/p.o) was significantly (p<0.05) and (p<0.01) decreased respectively. However glibenclamide (0.4mg/kg b.w/p.o) treatment showed significant (p<0.01) decrease when compared to Alloxan induced diabetic animals. Results are shown in Table 11 and Figure 11. Serum LDL-cholesterol The serum LDL-cholesterol level was significantly (p<0.01)increased in Alloxan induced diabetic rats when compared to control rats. Serum LDL-cholesterol level of diabetic rats treated with EEOT (200 and 400mg/kg b.wt/p.o) was significantly (p<0.05) and (p<0.01) decreased respectively. However glibenclamide (0.4mg/kg b.w/p.o) treatment showed significant (p<0.01) decrease when compared to Alloxan induced diabetic animals. Results are shown in Table 12 and Figure 12. Serum VLDL-cholesterol The serum VLDL-cholesterol level was significantly (p<0.01) increased in EEOT induced diabetic rats when compared to control rats. Serum VLDL-cholesterol level of diabetic rats treated with EEOT (200 and 400mg/kg b.wt/p.o)was significantly (p<0.05) decreased respectively. However glibenclamide (0.4mg/kg b.w/p.o) treatment showed significant (p<0.05) decrease when compared to Alloxan induced diabetic animals. Results are shown in Table 13 and Figure 13. Serum creatinine The serum creatinine level was significantly (p<0.01) increased in Alloxan induced diabetic rats when compared to control rats. Serum creatinine level of diabetic rats treated with EEOT (200 and 400mg/kg b.wt/p.o) was significantly (p<0.05) decreased respectively. However glibenclamide (0.4mg/kg b.w/p.o) treatment showed significant (p<0.01) decrease when compared to Alloxan induced diabetic animals. Result are shown in Table 14 and Figure 14. Table 3 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2723 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Table 4: Phytochemical screening of ethanolic extract of leaf of orthosiphon thymiflorus roth. S. no. Constituents Test Ethanolic extract 1. Alkaloids a) Mayer's reagent Absent b) Dragendorff's reagent Absent c) Hagner's reagent Absent d) Wagner’s reagent Absent 2. Carbohydrates a) Molisch's reagent Absent b) Fehling's solution A and B Absent c) Benedict's reagent Absent d) Barfoed's reagent Absent 3. Protein a) Biuret test Absent b) Millon's reagent Absent 4. Steroids a) Libermann's burchard test Present b) 5% potassium hydroxide Present 5. Phenols a) Ferric chloride Absent b) 10% Sodium chloride Absent 6. Tannins a) 10% Lead acetate solution Absent b) 10% Sodium chloride Absent c) Aqueous bromine solution Absent 7. Flavanoids a) Amyl alcohol + Sodium acetate + Present Ferric chloride b) Con. H2SO4 Present c) Magnesium turning test Present 8. Gums and Mucilage Swelling test Absent 9. Glycosides Glacial acetic acid + Ferric chloride + Con. Sulphuric acid Absent 10. Sterols 5 % Potassium Hydroxide Absent 11. Saponins Foam test Absent 12. Terpenes Tin + Thionyl chloride Present Table 5: Acute oral toxicity studies (OECD 423 guideline). S. Treatment Dose Weight of Signs Onset Reversible Duration No. group animal in gms of of or Before After toxicity toxicity irreversible test test No 1. EOT 2g/kg 160 170 signs of Nil Nil 14 days toxicity No 2. EOT 2g/kg 164 175 signs of Nil Nil 14 days toxicity No 3. EEOT 2g/kg 165 180 signs of Nil Nil 14 days toxicity www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2724 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Effect of EEOT extract of whole plant on blood glucose in alloxan induced diabetic rats [NG-OGTT] Blood glucose levels (mg/dl) Test Group 60min Sample s 0 min 30 min (glucose 120min 150min 270min (mg/kg) load) I Control 75.38±1.8 80.2±2.3 75.9±0.8 127.18±0.51 100.23±0.55 78.23±0.24 50.9±1.7 41.88±0.6 90.8±0.3 71.5±0.52 55.8±0.52 II Std-0.4 73.11±2.7 ** ** ** ** ** EEOT- 70.2±2.3 61.85±0.71 122.28±0.5 62.46±0.65 III 65.5±1.2 83.01±0.45* 200 ns * ns * EEOT- 72.01±0.8 59.03±0.27 110.15±0.5 75.95±0.5 60.6±0.52 IV 74.7±1.9 400 ns ** * ** ** The values are expressed as mean ± SEM. Statistical significance test was done by ANOVA followed by Dunnet’s test. The blood glucose values of group II, III and IV are compare with control animal values. *-p< 0.05, **-p< 0.01, ns-non significant. 150 I II 100 III IV 50 Blood glucose(mg/dl) Blood 0 0 min 30min 60 min120 min150 min270 min Time(min) Figure-5. Table 6: Effect of sub-acute treatment of EEOT on body weight changes in alloxan induced diabetic rats. Dose Body weight (gm) Group Treatment 1.(Kg -1 0 10th Day 15th Day bwt) th Day I Control(Distilled water) 5 ml 192.45±1.24 196.92 ± 1.2 201.5 ±1.5 II Disease control(Alloxan) 150mg 210.24±0.99 167.89±1.4** 153.5±0.7** III Standard(Glibenclamide+Alloxan) 0.4mg 183.13±2.64 185.47±3.2* 187.9±1.5** IV Test I (EEOT+Alloxan) 200mg 197.62 ± 4.3 166.6±5.4ns 173.2± 4.2* V Test II (EEOT+Alloxan) 400mg 189.92 ± 1.7 175.6 ±1.3* 187.6±4.1** www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2725 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The values are expressed as mean ± SEM. Statistical significance test was done by ANOVA followed by Dunnet’s test.The body weights of group II, III and IV are compared with control.*-p< 0.05, **-p<0.001, ns-non significant. 250 200 150 100 50 Body weight(gms) Body 0 0 10 15 Time(days) Group I. Group II Group III Group IV Group V Figure 6. Table 7: Effect of sub acute treatment of EEOT on blood glucose in Alloxan induced diabetic rats. Dose (Kg - Blood Glucose (mg/dl) Group Treatment 1 Body 0th Day 10th Day 15th Day Weight) I Control (distilled water) 5 ml 76.27±1.27 79.27±1.93 82.35±9.7 II Disease control (Alloxan) 150 mg 246.51±5.3 266.07±5.3** 289.42±5.23** III Standard 0.4mg 231.12±4.8 168.65±4.2b** 108.74±2.51** (Glibenclamide+Alloxan) IV Test I (EEOT+Alloxan) 200 mg 233±3.4 179.69±3.06* 132.67±4.1** V Test II (EEOT+Alloxan) 400mg 229.97±3.2 169.89±4.11** 118.24±4.67** The values are expressed as mean ± SEM. Statistical significance test for comparision was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with Group I .b-groups III, IV, V are compared with group. **P<0.01, *P<0.05 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2726 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 400 I II 300 III IV 200 V 100 Blood glucose(mg/dl) Blood 0 0 day 10 day 15 day Groups Figure 7 Table 8: Effect of EEOT on serum total protein in Alloxan induced diabetic rats. Dose Serum total protein Groups Treatment (kg-1body (mg/dl) weight) I Control(dis.water ) 5 ml 6.5593±0.1556 II Diabetic control(Alloxan) 150 mg 3.6388±0.0916** III Standard (Glibenclamide + Alloxan) 0.4mg 6.038±0.2600** IV Test I (EEOT +Alloxan) 200mg 5.1215±0.5387* V Test II (EEOT + Alloxan) 400mg 5.2698±0.7055** The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with Group I .b-groups III, IV, V are compared with group II **P<0.01,*P<0.05. 8 6 4 2 Total protein (mg/dl) protein Total 0 I II III IV V Groups Figure 08 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2727 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Table 09: Effect of EEOT total cholesterol in Alloxan induced diabetic rats. Dose(Kg - 1 Body Total Cholesterol Group Treatment Weight) (mg/dl) I Control (distilled water) 5 ml 116.505±3.8146 II Disease control (Alloxan) 150mg 167.43±3.0334** III Standard 0.4mg 121.422±3.119** (Glibenclamide+Alloxan) IV Test I (EEOT+Alloxan) 200mg 127.147±3.81** V Test II (EEOT+Alloxan) 400mg 141.77±4.397** The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with group I values, b- Group III, IV and V are compared with Group II. **P<0.01 200 150 100 50 Total cholesterol(mg/dl) Total 0 I II III IV V Groups Figure 9 Table 10: Effect of EEOT on HDL Cholesterol in Alloxan induced diabetic rats. Dose(Kg - 1 Body HDL-Cholesterol Group Treatment Weight) (mg/dl) I Control (distilled water) 5 ml 41.06±2.87 II Disease control (alloxan) 015mg 27.61±1.69** III Standard(Glibenclamide+Alloxan) 0.4mg 37.00±2.45* IV Test I (EEOT+Alloxan) 200mg 34.3±3.14* V Test II (EEOT+Alloxan) 400mg 39.60±2.47** The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with group I values, b- Group III,IV and V are compared with Group II. **P<0.01 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2728 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 50 40 30 20 10 HDL-Cholesterol(mg/dl) 0 I II III IV V Groups Figure 10 2. Table 11: Effect of EEOT on triglycerides in Alloxan induced diabetic rats. Dose (Kg - 1 Group Treatment Trigly Cerides (mg/dl) Body Weight) I Control (distilled water) 5ml 142.33±3.45 II Disease control (Alloxan) 45mg 188.36±3.46** III Standard (Glibenclamide+Alloxan) 0.4mg 151.85±3.02** IV Test I (EEOT+Alloxan) 250mg 168.1±3.44* V Test II (EEOT+Alloxan) 500mg 150.8±3.21** The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with group I values, b- Group III, IV and V are compared with Group II. **P<0.01 *-p<0.05 200 150 100 50 Triglycerides(mg/dl) 0 I II III IV V Groups Figure 11 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2729 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Table 12: Effect of EEOT on LDL-Cholesterol in Alloxan induced diabetic rats. Dose (Kg - 1 Body Group Treatment LDL (mg/dl) Weight) I Control (distilled water) 5 ml 48.00±4.58 II Disease control (Alloxan) 150 mg 102.18±4.99** III Standard(Glibenclamide+Alloxan) 0.4 mg 55.56±4.56** IV Test I (EEOT+Alloxan) 200 mg 83.75±3.15* V Test II (EEOT+Alloxan) 400 mg 55.46±3.08** The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test.a-Group II is compared with group I values, b- Group III, IV and V are compared with Group II. **P<0.01 *-P<0.05. 150 100 50 LDL(mg/dl) 0 I II III IV V Groups Figure 12 Table 13: Effect of EEOT on VLDL-Cholesterol in Alloxan induced diabetic rats. Group Treatment Dose (Kg - 1 Body VLDL(mg/dl) Weight) I Control (distilled water) 5 ml 28.47±0.63 II Disease control (Alloxan) 150 mg 37.51±0.97** III Standard(Glibenclamide+Alloxan) 0.4 mg 32.39±0.90* IV Test I (EEOT+Alloxan) 200 mg 33.87±0.70* V Test II (EEOT+Alloxan) 400 mg 32.56±0.65* The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with group I values, b- Group III, IV and V are compared with Group II. **P<0.01 *-p<0.05. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2730 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 40 30 20 VLDL(mg/dl) 10 0 I II III IV V Groups Figure 13 Table 14: Effect of EEOT whole plant extract on creatinine in Alloxan induced diabetic rats. Dose (Kg - 1 Body Group Treatment Creatinine (mg/dl) Weight) I Control (distilled water) 5 ml 0.72±0.061 II Disease control (Alloxan) 150 mg 1.72±0.05** III Standard(Glibenclamide+Alloxan) 0.4 mg 1.23±0.03* IV Test I (EEOT+Alloxan) 200 mg 1.36±0.02* V Test II (EEOT+Alloxan) 400 mg 1.28±0.04* The values are expressed as mean ± SEM. Statistical significance test for comparison was done by ANOVA, followed by Dunnet’s test. a-Group II is compared with group I values, b- Group III, IV and V are compared with Group II. **P<0.01 *-p<0.05 2.0 1.5 1.0 0.5 Creatinine(mg/dl) 0.0 I II III IV V Groups Figure 14 www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2731 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Histopathological analysis of pancreas (40 x) Figure No. Report 15.1 H & E stained section shows pancreas with Normal control normal islets and acinar cells. 15.2 H & E stained section shows damaged and Diabetic control atrophic islet with acni. 15.3 H & E stained section shows preserved Glibenclamide treated pancreatic islet cells. 15.4 H & E stained section shows small islet cells EEOT 200 mg/kg body weight 15.5 H & E stained section shows hyperplastic islet EEOT 400 mg/kg body weight with acni Fig. 15.1: (Normal control) 40x Fig. 15.2: (Diabetic control) 40x Fig. 15.3: (Glibenclamide treated) 40x. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2732 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences Fig. 15.4: (EEOT 200 mg treated) 40x. Fig. 15.5: (EEOT 500 mg treated) 40x. VII. DISCUSSION Plants have been used as source of drugs for the treatment of diabetes mellitus in developing countries where the cost of conventional medicines represents a burden to the population. Many species have been reported to present antidiabetic activity.[45] Working on the same line, we have undertaken a study on Orthosiphon thymiflorus Roth for its antidiabetic property along with its anti oxidant potential. Preliminary phytochemical analysis of the EEOT extract of whole plant showed that the plant has a rich possession of phytochemicals like flavonoids and steroids. Acute oral toxicity studies reveal that EEOT whole plant extract did not produce any mortality or signs of toxicity at the dose of 2000 mg/kg b.w. p.o, in experimental rats. The EEOT whole plant extract at doses 200 and 400 mg/kg bw.po.did not significantly suppress blood glucose levels in over night fasted normoglycaemic animals but showed significant improvement in glucose tolerance in glucose fed hyperglycaemic normal rats. Such an effect may be accounted for, in part, by a decrease in rate of intestinal glucose absorption, achieved by an extra pancreatic action including stimulation of peripheral glucose utilization or enhancing glycolytic and glycogenic process.[46] Alloxan is the most commonly employed agent for the induction of experimental diabetic animal models of human insulin dependent diabetes mellitus.[47] There is an increasing evidence that alloxan causes diabetes by rapid depletion of β-cells, by DNA alkylation and accumulation of cytotoxic free radicals that is suggested to result from initial islet inflammation, followed by infiltration of activated macrophages and lymphocyte in the inflammatory focus.[48] www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2733 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences In the sub-acute study, glebenclamide treatment brought down the sugar levels from the first day of the treatment. EEOT 200 mg and 400mg treatment produces significant reduction in blood glucose levels from 10th of treatment and a steady decrease was observed there after. Another possibility for the activity may be due to presence of phytochemicals like flavanoids, phenolics and alkaloids etc.[53] Histopathological studies that showed prominent islets cell hyperplasia and regeneration of islet cell show a proof for the possible antidiabetic property of the leaf extract of Orthosiphon thymiflorus Roth. Lipids play an important role in the pathogenesis of diabetes mellitus. The level of serum lipids is usually raised in diabetic condition and such an elevation poses to be a risk factor for cardiovascular diseases like coronary heart disease and two to four fold risk for diabetes. Atherosclerosis which constitutes the main cause of morbidity and mortality in diabetes mellitus.[54] In the present study an elevated serum total cholesterol and reduced HDL- cholesterol, triglycerides, LDL-cholesterol, VLDL-choleseterol, creatinine was observed in Alloxan-induced diabetic rats. The glibenclamide treatment, EEOT 200mg and EEOT 500 mg treatment in diabetic animals produced beneficial improvement in the lipid profile. VIII. SUMMARY AND CONCLUSION Orthosiphon thymiflorus Roth is a plant traditionally used for the treatment of diabetes mellitus. The claim for the utility of the plant in the treatment of diabetes has not been scientifically evaluated. This dessertion was designed based in the traditional claim to emphasize the antihyperglycaemic, antioxidant potential of the ethanolic extract of leaf of Orthosiphon thymiflorus Roth. Preliminary phytochemical analysis of the EEOT extract of leaf of the plant showed that the plant has a rich possession of phytochemicals like flavonoids and steroids. Acute oral toxicity studies reveal that EEOT did not produce any mortality or signs of toxicity at the dose of 2000 mg/kg b.w.p.o, in experimental rats. Treatment of EEOT shown moderate hypoglycaemic effect on normal animals and significant improvement in glucose tolerance on glucose fed hyperglycaemic rats. In the sub acute study a steady decrease in blood glucose level was observed on EEOT treatment in Alloxan induced diabetic rats. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2734 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences The treatment of EEOT showed marked increase in body weight, marked increase in protein, HDL cholesterol levels in serum of Alloxan induced diabetic animals. At the same time significant decrease in total cholesterol, LDL-cholesterol, VLDL-cholesterol, triglycerides and creatinine levels was observed in serum of diabetic animals. Based on the obtained results and observations, we can infer that the leaf of Orthosiphon thymiflorus Roth could be used for the supportive treatment of diabetes mellitus, as the plant also offers effective protection against free radicals that form the basis for the development of diabetic complications. Further studies are required to establish the anti hyperglycaemic activity of Orthosiphon thymiflorus Roth in terms of molecular mechanism(s) involved in the activity. REFERENCES 1. Adeneye AA, Amole OO, Adeneye AK, Hypoglycaemic and hypocholesteromic activities of the aqueous leaf and seed extract of phyllanthus amarus in mice, Ftoterapia, 2006; 77: 511-514. 2. Rema dheeer, Nema RK, Manoj dheer, Pradeepbhatnagar, Healing power of herbs:Let us not the antagonize it, Planta indica,2006; 4: 1-4. 3. Maiti R, Jana D, Das UK, Ghosh D, Antiidabetic efect of aqueous extract of seed of Tamarindus indica in Streptozotocin-induced diabetic rats,Journal of ethnopharmacology, 2004; 92: 85-91. 4. Achyut Narayan Kesari, Rajesh kumar gupta,Santosh Kumar Singh, Hypoglycaemic and antihyperglycaemic activity of Aegle marmelos seed axtract in normal and diabetic rats, Journal of Ethnopharmacology,2006; 107: 374-379. 5. Chakrabarti S, Kanti Biswas T, Tapan S, Begum R. Antidiabetic activity of Caesalpinia bonducella F. in chronic type II diabetic model in Long-Evans rats and evaluation of insulin secretagogue property of its fractions on isolated islets, Journal of Ethnopharmacology, 2005; 97: 117-122. 6. GyorgyJ, Can type 2 diabetes mellitus be considered preventable?,Diabetes Research andClinical Practice, 2005; 68S1: S73-S81. 7. Kavitha Jv, Joseph F, Rosario, Chandran J .Hypoglycaemic and other related effect of Boswelia glabra in alloxan-induced diabetic rats,Indian journal of physiology and pharmacology, 2007; 51(1): 29-39. 8. Vinay Kumar, Abbas K, Nelson F, Pathologic Basis of Disease, 7: 1189-1207. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2735 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 9. Badole S, Patel N, Bodhankar S, Jain B, Antihyperglycaemic activity of aqueous extract of leaves of Cocculus hirsutus(L) Diels in alloxan-induced diabetic mice, 2006; 38: 49- 53. 10. Nicholas A Boon, Colledge, Walker R. Davson’s Principle & Practice of Medicine Elsveir, 14: 806-846. 11. Kannur DM, Hukkeri VI Akki K. Antidiabetic activity of Caesalpinia bonducella seed extracts in rats, 2006; 77: 546-549. 12. Vijayakumar M, Govindarajan R, Rao G, Action of Hybrophila auriculata against streptozotocin-induced oxidative stress,Journal of Ethnopharmacology, 2006; 104: 356-361. 13. Srinivasan R, Chandrasekar MJN, Nanjan MJ, Suresh B. Antioxidant activity of Caesalpinia digyna root, 2007; 1-8. 14. Arthur C, Guyton. Text book of medical physiology, Saunders Company, 855-867. 15. Sy GY, Cisse A, Nogonierma RB, Sarr M, Mbdj NA, Hypoglycaemic and antidiabetic activity of acetonic extract of Vernonia colorata leaves in normoglycaemic alloxan- induced diabetic rats, Journal of Ethnopharmacology, 2005; 98: 171-175. 16. Hyun Park S, Kwon Ko S, Hyun Chung S. Euonymus alatus prevents the hyperglycaemia and hyperlipidemia induced by high-fat diet in ICR mice, 2005; 102: 326-335. 17. Sutharason L, Hariharan RS, Vamsadhara Drug utilization study in diabetology outpatieny setting of a tertiary hospital, Indian journal of pharmacology, 2003; 35: 237-240. 18. Rang HP, Dale MM, Ritter JM, Moore PK, Pharmacology, Churchill Livingstone,Elsvier limited.2003; 7: 380-385. 19. Laurence L, Goodman &Gilman’s, The Pharmacological Basis of THERAPEUTICS, McGRAW-HILL, 2005; 4: 1613-1646. 20. Sathoskar, Kale, Bhandarkar’s Pharmacology and Pharmacotherapeutics, Popular prakashan, 2000; 7: 901. 21. Chattopadhyay S, Ramanathan M, Das j and Bhattacharya SK. Animal models in experimental diabetes. Indian journal of Experimental Biology, 1997; 35: 1141-1145. 22. Sigurd lenzen; Alloxan and streptozotocin diabetes 23. Mustafa A, David EL, Diabetes, oxidative stress and physical exercise, Journal of sports science and medicine, 2002; 1: 1-14. 24. Tiwari A.K. Antioxidants: New generation therapeutic base for treatment of polygenic disorders, Current Science, 2004; 86: 1092-1102. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2736 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 25. Deshpande SS, Madhavi DL and Salunkhe DK. Nutritional and health aspects in food antioxidants. In: Technological, Toxicological and Health perspectives. Marcel Dekker.Inc, New York, 1995; 371-391. 26. Halliwell B and Gutteridge JMC. Free radicals in Biology and Medicine Edition. Oxford: Clarendron Press, 1989. 27. Ray G and Hussain SA, Oxidants, antioxidants and carcinmogenesis. Indian Journal of Experimental Biology, 2002; 41: 1213-1232. 28. Devasagayam TPA, Boloor KK and Ramasarma T. Methods for estimating lipid peroxidation:an analysis of merits and demerits. Indian journal of Biochemistry and Biophysics, 2003; 40: 300-308. 29. Akhar MS, Khan MA, Malik MT, Hypoglycaemic activity of Alpina galangal rhizome and its extracts in rabbits, Fitoterapia, 2002; 73: 623-628. 30. Santhakumari P, Prakasam A, Pugalendi KV, Modulation of oxidative stress parameters by treatment with piper betle leaf in streptozocin induced diabetic rats, Indian journal of Pharmacology, 2003; 35: 373-378. 31. http://ayurvedicmedicinalplants.com/plants/3755.html 32. http://en.wikipedia.org/wiki/Orthosiphon 33. http://zipcodezoo.com/Plants/O/Orthosiphon_thymiflorus/ 34. S.Sundarammai; R.Thirugnanasampandam; M. Tamil Selvi.Chemical composition analysis and antioxidant activity evaluation of essential oil from Orthosiphon thymiflorus (Roth) Sleesen.Asian Pasific Journal Tropical Biomedicine, 2012; S112-S115. 35. Sundaram R, Mitra SK, Antioxuidant activity of caesalpinia digyin in vitro and in vivo experimaental models, The Antiseptic, 2006; 103: 522-528. 36. Chaudary GR, Sharma VN, Dhar ML, Chemical examination of the roots of Caesalpinia digyna,Journal of scientific and industrial research, 1954;13:147-148. 37. Kokate Ck, Practical Pharmacognosy, Edition, Vallabh Pakasham, Delhi, 1991; 5: 107-121. 38. The Ayurvedic Pharmacopoea of India Part-1, Govt of india, Ministry of healthy& family Welfare. New Delhi, 15-16. 39. Ecobichon DJ, The basis of Toxicity testing, CRC press, NewYork, 1997; 2: 43-88. 40. Latha M, Pari L, Sandhya S, Ramesh B, Insulin secretagogue activity and cytoprotective role of the traditional antidiabetic plant scoparia dulcis, Life sciences, 2004; 75: 2007- 2014. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2737 Mariyam Begum. World Journal of Pharmacy and Pharmaceutical Sciences 41. Somani R, Sanjay K, Singhai AK, Antidiabetic potential of butea monosperma, in rats, Fitoterapia, 2006; 77: 80-90. 42. Mustafa A, Didem D, Orhan N, Invivo antidiabetic and antioxidant potential of Helichrysum plicatum ssp.plicatum capitulums in streptozotocin-induced-diabetic rats, Jouanal of Ethanopharmacology, 2007; 109: 54-59. 43. Annie S, Rajendran K, Rakesh B, Effect of aqueous bark extract of Garuga pinnata Roxb. In streptozocin –nicotinamide induced type-II diabetic mellitus, Journal of Ethanopharmacology, 2006; 107: 285-290. 44. Muruganandam S, Srinivasan K, Gupta S, Gupta PK, Effect of magniferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats, Journal of Ethanopharmacology, 2005; 97: 497-501. 45. Singhavi AK, Aherwar B, Effect of terminalia chebula fruits on Lipid profile of rats, Journal of Natural Remedies, 2003; 3: 31-35. 46. Madhu CG, Dharan PC, Devi BB, Effect of vitamin E supplementation on diabetes induced oxidative stress in experimental Diabetes in rats, Indian Journal of Experimental Biology, 2005; 43: 177-180. 47. Oliveria ACP, Denise CE, Luiz ASA, Effect of the extracts and fractions of Baccharistrimeria and Syzygium cumini, on glycaemia of diabetic and non-diabeticmice, Journal of Ethanopharmacology, 2005; 102: 465-469. 48. Mukherjee PK, Saha K, Pal M, Saha BP, Effect of Nelumbo nucifera rhizome extract on blood sugar level in rats. Journal of Ethanopharmacology, 1997; 58: 207-213. 49. Eddoku M, Maghrani M, Michel JB, Hypoglycemic effect of triticum repens, in normal and diabetic rats, Journal of Ethanopharmacology, 2005; 102: 228-232. 50. Jung CH, Seog HM, Ehoi W, Choi HD, Effect of wild ginseng leaves on lipidperoxidation levels and antioxidant enzyme activities in streptozotocin diabetic rats, Journal of Ethanopharmacology, 2005; 98: 245-250. 51. Kameswara rao B, Kesavalu MM, Apparao C, Evaluation of antidiabetic effect of momordica cymbalaria fruit in alloxan diabetic rats, 2003; 74: 7-13. 52. Ronald k, George K, Allas N, Elsiver Publication, 14: 12. 53. Sniderman AD, Bhopal R, Prabhakaran D, Sarrafzadegan and Tchernof ,why might soufa Asians be so susceptible to central obesity and its atherogenic consequences The adipose tissue overflow hypothesis‖. International Journal of Epidemiology, 2007; 36: 220-225. 54. Al- shamaony, Shabba M, Hypoglycemic effect of arteminissia herba aba, Journal of Ethanopharmacology, 1994; 2: 167-171. www.wjpps.com │ Vol 10, Issue 8, 2021. │ ISO 9001:2015 Certified Journal │ 2738