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The foliages of Lagerstreomia speciosa were collected in August to September of 2010 from different locations of Rawalpindi and Islamabad. The works stuffs ( foliages ) 12Kg were cleaned with distilled H2O and shadiness dried at room temperature and so powdered.

Alloxan monohydrate, Ethanol Commercial, Methanol, Diphenyl-1-picrylhydrazyl ( DPPH ) , Folin Ciocalteu ‘s reagent ( Phosphomolybdate and Phosphotungstate ) , Ascorbic acid, Normal saline, Distilled H2O, Gallic Acid monohydrate. All these chemicals were purchased from Chief Scientific, Rawalpindi, Pakistan.

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2.3 Equipments:

Rotary vacuity evaporator, UV-Vis Spectrophotometer, Glucometer, Balance, coney coops, coney holders, Blood aggregation tubings, panpipes ( 5ml, 26mm Needle gage ) were used.

2.4 PREPARATION OF EXTRACTS:

The collected foliages ( 12Kg ) were shade dried, coarsely powdered and extracted with 50 % ethyl alcohol ( 100g/1L ) by cold maceration procedure. The infusion was filtered and concentrated in vacuity and kept in vacuity desiccators for complete remotion of solvent [ 17 ] .

Thin Layer Chromatography ( TLC ) of the L. speciosa infusion was run one dimensionally in the nomadic stage dissolver ( ethyl ethanoate – ethanol – H2O, 5:1:5, v/ v/ V ) at room temperature of 20-25°C. The concentrated infusion was spotted on the lower left of the TLC home base and the diameter of the topographic point in each chromatogram was usually about 5mm. Authentic markers of flavonol ( quercetin ) and flavonoid glycoside ( rutin ) obtained commercially were co-chromatographed. Designation of the flavonoids in the infusions was identified under UV visible radiation after the application of ammonium hydroxide ( Adam et al. , 2002 ; Guorong Fan et al. , 2006 ) .

Trials for Alkaloids:

The presence of alkaloids in L. speciosa infusion was confirmed by assorted trials e.g.

1. Mayer ‘s Trial: Specimens with Mayer ‘s reagent give Cream or pale xanthous ppt.

2. Dragendroff Reagent Trial: Specimens with Dragendroff Reagent give orange ppt.

3. Otto wagners Test: Specimens with Wagner ‘s Reagent give brown or reddish brown ppt.

Trials for Glycosides:

The presence of glycosides in L. speciosa infusion was confirmed by assorted trials e.g.

1. Borntrager ‘s Trial: Specimens with Borntrager ‘s reagent give ruddy pink colour.

2. Modified Borntrager ‘s Trial: Specimens with Modified Borntrager ‘s reagent spring pink colour.

3. Bromine Trial: Specimens with Bromine trial gives pale xanthous ppt.

4. Azotic Acid Test: Specimens with Nitric Acid trial gives brown colour.

The hydro alcoholic infusion showed presence of flavonoids, alkaloids and glycosides.

Adam JH, Ramian Omar, Wilcock CC. Phytochmeical showing of flavonoids in three loanblends of Napenthes ( Napenthaceae ) and their putative parental species from Sarawak and Sabah. Online J biol sci. 2002 ; 2 ( 9 ) :623-625

Guorong Fan, Jinyong Peng, Yutian Wu. ; Preparatory Separation and Isolation of Three Flavonoids and Three Phloroglucinol Derivatives from Hypericum japonicum Thumb utilizing High-Speed Countercurrent Chromatography by Stepwise Increasing the Flow Rate of the Mobile Phase. J Liq Chrom Tech, 2006 ; 29: 1619-1632

INVITRO STUDIES

2.6 TOTAL PHENOLIC COMPOUND ESTIMATION

Antioxidant compounds by and large contain phenolic group ( s ) and therefore, the sum of phenolic compounds in the infusion of foliages was estimated by utilizing Folin-Ciocalteu ‘s Reagent [ 18 ] . In a series of trial tubings, 0.4 milliliter of the infusion in Methanol was taken, assorted with 2 milliliters of Folin-Ciocalteu ‘s reagent and 1.6 milliliter of Sodium Carbonate. After agitating, the reaction mixture was kept for 2 hours. The optical density measured at 750 nanometers utilizing a Shimadzu UV-160 Spectrophotometer. Standard curve was prepared and one-dimensionality was obtained in the scope of 2.5 to 25 I?g/ml, by utilizing Gallic Acid Monohydrate. Using the criterion curve the entire phenolic compounds content was calculated and expressed as Gallic Acid Equivalent in mg/g or % w/w of the infusion. [ 17 ]

17. Pareek Anil, Suthar Manish, Rathore Garvendra S. , Bansal Vijay, Kumawat Tarachand: In Vitro Antioxidant Studies of Lagerstroemia speciosa Leaves.

18. Sadasivam S, Manikam A. Wiley Eastern Limited. New Delhi. 1992: 187

2.7 ANTIOXIDANT POTENTIAL

2.7.1 SCAVENGING EFFECTS OF PLANT ON DPPH RADICAL

Free extremist scavenging consequence was determined utilizing the free extremist generator DPPH ( diphenyl-1-picrylhydrazyl ) .

Different concentrations of works infusion were prepared in methyl alcohol runing from 25I?g/mL to 250I?g/mL.

Standard DPPH solution incorporating 400micromole DPPH was prepared in methyl alcohol.

Standard DPPH solution was so assorted with test drug dilution at a ratio 1:3 i.e. 1mL of trial infusion was assorted with 3mL of Standard DPPH solution in different decently closed containers. The mixtures were kept in the dark at a room temperature for 90 proceedingss.

Optical density of ensuing solution was measured utilizing spectrophotometer at 517 nanometer. Scavenging activity was calculated by utilizing equation: [ 19-23 ]

Ascorbic Acid was used as a criterion antioxidant

Scavenging activity ( % ) =

Optical density of sample at 517 nm / Absorbance of control at 517 nanometers

The antioxidant activity is expressed as IC50. The IC50 value is the step of concentration in I?g/ml of infusion that inhibits 50 % of DPPH groups.

19. Budge A.J. and Aust S.D. , Microsomal lipid peroxidation, : Methods in Enzymology, 1978, 52, 302.

20. Dorman H.J.D. , Peltoketo A. , Hiltunen R. and Tikkanen M.J. : Word picture of the antioxidant belongingss of de-odourised aqueous infusions from selected Lamiaceae herbs, Food Chemistry, 2003, 83, 255.

21. Duh P.D. , Yen G.C. , Yen W.J. , Wang B.S. and Chang L.W. : Effectss of puerh tea on oxidative harm and azotic oxide scavenging, Journal of the American Oil Chemistry Society, 2004, 52, 8169.

22. Bowry V.W. , Ingold K.U. and Stocker R. : Vitamin E in human low-density lipoprotein – when and how this antioxidant becomes a pro oxidizer, Biochemistry Journal, 1992, 288, 341.

23. Hsieh C.L, Lin Y.C, Ko W.S, Peng C.H, Huang C.N. and Peng R.Y ; Inhibitory consequence of some selected nutraceutic herbs on LDL glycation induced by glucose and glyoxal, Journal of Ethnopharmacology, 2005, 102, 357

INVIVO STUDIES IN RABBITS

2.8 Experimental Animals:

White coneies with mean weight 1.5 Kg were used. The animate beings were housed in a good aerated room with controlled lighting. All animate beings were kept at the carnal house of National Institute of Health Islamabad. Animals were housed in chromium steel coops under standard research lab status ( light period 8.00a.m to 8.00p.m 23A±2 oC, comparative humidness 55 % , green fresh fish and H2O was available. Animals received human attention. For at least 3 yearss before start of survey coneies were acclimatized to the Laboratory environment. The survey protocols were approved by the N.I.H ethical commission and the experiments were carried out after acquiring blessing of the protocols from the Research Committee at Riphah Institute of Pharmaceutical Sciences, Riphah International University ( RIPS, RIU ) – Capital of pakistan, Pakistan.

2.9 INDUCTION OF DIABETES IN RABBITS:

The purpose of this experiment was to bring on diabetes in coneies with the aid of Alloxan monohydrate at the dosage of 300 mg/kg/day for three yearss in divided doses. Alloxan was administered in divided doses to avoid deceases in coneies. Blood glucose degree and organic structure weights were noted ab initio.

The animate beings normally show following marks of the diabetes: Asthenia i.e. , failing because ability to utilize glucose as an energy beginning, Polydipsia i.e. , unnatural thirst, Polyuria i.e. , addition in volume of piss, Weight loss because of thin organic structure mass, desiccation because coney will seek to acquire rid of extra blood glucose. Initiation of diabetes was confirmed by mensurating the blood glucose degree.

At the terminal of 10 yearss, a rise in blood sugar was noticed in the group, which was compared with the standard normal blood glucose value in Rabbits i.e. 75-100 mg/dL. Rabbits with blood glucose degree 125 mg/dl and above were selected.

2.10 TREATMENT Agenda:

The coneies ( n = 6 per group ) were divided in 4 groups. After 10 yearss of intervention with Alloxan, the coneies demoing stabilized diabetes holding fasting blood glucose ( FBG ) values 125mg/dl or above was considered as diabetic animate beings consider it as nothing twenty-four hours. Dosing with the herbal infusions was started on the first twenty-four hours and continued for 30 yearss harmonizing to the undermentioned agenda:

Sr. No.

Treatment

Dose

Group1

Normal Control

Distilled H2O

Group2

Diabetic Control

Distilled Water

Group3

Diabetic ( Glibenclamide )

0.6mg/Kg organic structure weight

Group4

Diabetic ( L. speciosa )

100 mg/Kg organic structure weight

2. 11 BLOOD Sampling:

Blood samples were taken from fringy ear vena of the coneies by utilizing 26 mm guage acerate leaf after 0, 15 and 30 yearss for haemolytic parametric quantities such as

Blood glucose degrees,

Lipid profile i.e. , Serum Cholesterol, Serum Triglycerides, Serum HDL and Serum LDL, and

LFTs ( AST, ALT and Alk-Phosphatase ) .

Above blood trials were carried out in coaction with Riphah Diagnostic & A ; Research Lab at Islamic International Medical College Hospital, Islamabad, Pakistan on payment.

IN-VIVO STUDIES IN HUMANS

The purpose of this experiment was to analyze anticholesterolemic consequence of Lagerstroemia speciosa in healthy voluntaries holding Body Mass Index ( BMI ) greater than 24.9. BMI is a utile step of corpulence and fleshiness. [ 24 ] The BMI values for scraggy, normal weight, corpulence and corpulent persons are given below ;

Underweight if BMI & lt ; 18.5

Normal weight if BMI 18.5 to 24.9

Overweight if BMI 25 to 29.9

Fleshiness if BMI is of 30 or greater

24. hypertext transfer protocol: //www.nhlbi.nih.gov/health/public/heart/obesity/lose_wt/risk.htm

INCLUSION CRITERIA

The choice of voluntaries was carried out carefully. Subjects were

Having age from 18 – 55 old ages

Sing in good wellness based on medical history, physical scrutiny. Volunteers after complete blood count, lipid profile and liver map trial were entitled to take part in this survey.

EXCLUSION CRITERIA

Subjects were excluded if any had:

Abnormal findings upon medical histories, physical scrutinies and testing trials.

Known positive human immunodeficiency virus ( HIV ) serology, AIDS.

Pregnant or nursing or had donated blood within 30 yearss prior to analyze.

Taken any antihyperlipidemic drugs for one hebdomad before survey and during full survey period.

The topics who had high blood pressure, hepatic or nephritic disease, engaged in heavy exercising

Limited mental capacity to extent, the topic will be unable to supply the legal consent and information sing side effects or tolerance to analyze drug.

EXPERIMENTAL DESIGN

Thirty healthy human voluntaries ( twenty = Male, xx = Female ) participated in the survey. Healthy voluntaries were recruited on the footing of their Body Mass Index ( & gt ; 24.9 ) . Written informed consent was obtained from each topic before beginning of survey.

Subjects were refrained from taking unwritten hypoglycaemic medicine and/or anticholesterolemic drugs at least 1 hebdomad prior to the survey. All topics were requested to keep High fat diet during the survey. Volunteers were divided into two groups.

Group 1 consists of 15 voluntaries who were administered Lagerstroemia speciosa tea ( One Table Spoon dry herb per cup ) three times a twenty-four hours for 2 hebdomads and this served as survey group.

Group 2 consists of 15 voluntaries who were administered field H2O three times a twenty-four hours for 2 hebdomads and this served as a control group.

BLOOD Sampling:

Blood samples were taken from voluntaries after 0, 15 and 30 yearss for haemolytic parametric quantities such as

Blood glucose degrees

Lipid profile i.e. , Serum Cholesterol, Serum Triglycerides, Serum HDL and Serum LDL, and

LFTs ( AST, ALT and Alk-Phosphatase )

Above blood trials were carried out in coaction with Riphah Diagnostic & A ; Research Lab at Islamic International Medical College Hospital, Islamabad, Pakistan on payment.

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