Leaves Of Lagerstreomia Speciosa Biology Essay

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The leaves of Lagerstreomia speciosa were collected in August to September of 2010 from different locations of Rawalpindi and Islamabad. The plant materials (leaves) 12Kg were cleaned with distilled water and shade dried at room temperature and then powdered.

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

2.3 EQUIPMENTS:

Rotary vacuum evaporator, UV-Vis Spectrophotometer, Glucometer, Balance, rabbit cages, rabbit holders, Blood collection tubes, syringes (5ml, 26mm Needle gauge) were used.

2.4 PREPARATION OF EXTRACTS:

The collected leaves (12Kg) were shade dried, coarsely powdered and extracted with 50% ethanol (100g/1L) by cold maceration process. The extract was filtered and concentrated in vacuum and kept in vacuum desiccators for complete removal of solvent [17].

Thin Layer Chromatography (TLC) of the L. speciosa extract was run one dimensionally in the mobile phase solvent (ethyl acetate - ethanol - water, 5:1:5, v/ v/ v) at room temperature of 20-25°C. The concentrated extract was spotted on the lower left of the TLC plate and the diameter of the spot in each chromatogram was normally about 5mm. Authentic markers of flavonol (quercetin) and flavonoid glycoside (rutin) obtained commercially were co-chromatographed. Identification of the flavonoids in the extracts was identified under UV light after the application of ammonia (Adam et al., 2002; Guorong Fan et al., 2006).

Tests for Alkaloids:

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The presence of alkaloids in L. speciosa extract was confirmed by various tests e.g.

1. Mayer's Test: Specimen with Mayer's reagent give Cream or pale yellow ppt.

2. Dragendroff Reagent Test: Specimen with Dragendroff Reagent give orange ppt.

3. Wagners Test: Specimen with Wagner's Reagent give brown or reddish brown ppt.

Tests for Glycosides:

The presence of glycosides in L. speciosa extract was confirmed by various tests e.g.

1. Borntrager's Test: Specimen with Borntrager's reagent give reddish pink color.

2. Modified Borntrager's Test: Specimen with Modified Borntrager's reagent give pink color.

3. Bromine Test: Specimen with Bromine test gives pale yellow ppt.

4. Nitric Acid Test: Specimen with Nitric Acid test gives brown color.

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

Adam JH, Ramian Omar, Wilcock CC. Phytochmeical screening of flavonoids in three hybrids 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.; Preparative Separation and Isolation of Three Flavonoids and Three Phloroglucinol Derivatives from Hypericum japonicum Thumb using 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 generally contain phenolic group(s) and hence, the amount of phenolic compounds in the extract of leaves was estimated by using Folin-Ciocalteu's Reagent [18]. In a series of test tubes, 0.4 ml of the extract in Methanol was taken, mixed with 2 ml of Folin-Ciocalteu's reagent and 1.6 ml of Sodium Carbonate. After shaking, the reaction mixture was kept for 2 hours. The absorbance measured at 750 nm using a Shimadzu UV-160 Spectrophotometer. Standard curve was prepared and linearity was obtained in the range of 2.5 to 25 μg/ml, by using Gallic Acid Monohydrate. Using the standard curve the total phenolic compounds content was calculated and expressed as Gallic Acid Equivalent in mg/g or % w/w of the extract. [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 radical scavenging effect was determined using the free radical generator DPPH (diphenyl-1-picrylhydrazyl).

Different concentrations of plant extract were prepared in methanol ranging from 25μg/mL to 250μg/mL.

Standard DPPH solution containing 400micromole DPPH was prepared in methanol.

Standard DPPH solution was then mixed with test drug dilution at a ratio 1:3 i.e. 1mL of test extract was mixed with 3mL of Standard DPPH solution in different properly closed containers. The mixtures were kept in the dark at a room temperature for 90 minutes.

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Absorbance of resulting solution was measured using spectrophotometer at 517 nm. Scavenging activity was calculated by using equation: [19-23]

Ascorbic Acid was used as a standard antioxidant

Scavenging activity (%) =

Absorbance of sample at 517 nm / Absorbance of control at 517 nm

The antioxidant activity is expressed as IC50. The IC50 value is the measure of concentration in μg/ml of extract that inhibits 50% of DPPH radicals.

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.: Characterisation of the antioxidant properties of de-odourised aqueous extracts 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.: Effects of puerh tea on oxidative damage and nitric 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 oxidant, 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 effect 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 rabbits with average weight 1.5 Kg were used. The animals were housed in a well aerated room with controlled lighting. All animals were kept at the animal house of National Institute of Health Islamabad. Animals were housed in stainless cages under standard laboratory condition (light period 8.00a.m to 8.00p.m 23±2 oC, relative humidity 55%, green fodder and water was available. Animals received human care. For at least 3 days before start of study rabbits were acclimatized to the Laboratory environment. The study protocols were approved by the N.I.H ethical committee and the experiments were carried out after getting approval of the protocols from the Research Committee at Riphah Institute of Pharmaceutical Sciences, Riphah International University (RIPS, RIU) - Islamabad, Pakistan.

2.9 INDUCTION OF DIABETES IN RABBITS:

The aim of this experiment was to induce diabetes in rabbits with the help of Alloxan monohydrate at the dose of 300 mg/kg/day for three days in divided doses. Alloxan was administered in divided doses to avoid deaths in rabbits. Blood glucose level and body weights were noted initially.

The animals usually show following signs of the diabetes: Asthenia i.e., weakness because ability to use glucose as an energy source, Polydipsia i.e., abnormal thirst, Polyuria i.e., increase in volume of urine, Weight loss because of lean body mass, dehydration because rabbit will try to get rid of excess blood glucose. Induction of diabetes was confirmed by measuring the blood glucose level.

At the end of 10 days, 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 level 125 mg/dl and above were selected.

2.10 TREATMENT SCHEDULE:

The rabbits (n = 6 per group) were divided in 4 groups. After 10 days of treatment with Alloxan, the rabbits showing stabilized diabetes having fasting blood glucose (FBG) values 125mg/dl or above was considered as diabetic animals consider it as zero day. Dosing with the herbal extracts was started on the first day and continued for 30 days according to the following schedule:

Sr. No.

Treatment

Dose

Group1

Normal Control

Distilled water

Group2

Diabetic Control

Distilled Water

Group3

Diabetic (Glibenclamide)

0.6mg/Kg body weight

Group4

Diabetic (L. speciosa)

100 mg/Kg body weight

2. 11 BLOOD SAMPLING:

Blood samples were taken from marginal ear vein of the rabbits by using 26 mm guage needle after 0, 15 and 30 days for hemolytic parameters such as

Blood glucose levels,

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

LFTs (AST, ALT and Alk-Phosphatase).

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

IN-VIVO STUDIES IN HUMANS

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The aim of this experiment was to study anticholesterolemic effect of Lagerstroemia speciosa in healthy volunteers having Body Mass Index (BMI) greater than 24.9. BMI is a useful measure of overweight and obesity. [24] The BMI values for underweight, normal weight, overweight and obese individuals are given below;

Underweight if BMI < 18.5

Normal weight if BMI 18.5 to 24.9

Overweight if BMI 25 to 29.9

Obesity if BMI is of 30 or greater

24. http://www.nhlbi.nih.gov/health/public/heart/obesity/lose_wt/risk.htm

INCLUSION CRITERIA

The selection of volunteers was carried out carefully. Subjects were

Having age from 18 - 55 years

Considering in good health based on medical history, physical examination. Volunteers after complete blood count, lipid profile and liver function test were entitled to participate in this study.

EXCLUSION CRITERIA

Subjects were excluded if any had:

Abnormal findings upon medical histories, physical examinations and screening tests.

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

Pregnant or nursing or had donated blood within 30 days prior to study.

Taken any antihyperlipidemic drugs for one week before study and during entire study period.

The subjects who had hypertension, hepatic or renal disease, engaged in heavy exercise

Limited mental capacity to extent, the subject will be unable to provide the legal consent and information regarding side effects or tolerance to study drug.

EXPERIMENTAL DESIGN

Thirty healthy human volunteers ( xx = Male, xx = Female) participated in the study. Healthy volunteers were recruited on the basis of their Body Mass Index (>24.9). Written informed consent was obtained from each subject before commencement of study.

Subjects were refrained from taking oral hypoglycemic medication and/or anticholesterolemic drugs at least 1 week prior to the study. All subjects were requested to maintain High fat diet during the study. Volunteers were divided into two groups.

Group 1 consists of 15 volunteers who were administered Lagerstroemia speciosa tea (One Table Spoon dry herb per cup) three times a day for 2 weeks and this served as study group.

Group 2 consists of 15 volunteers who were administered plain water three times a day for 2 weeks and this served as a control group.

BLOOD SAMPLING:

Blood samples were taken from volunteers after 0, 15 and 30 days for hemolytic parameters such as

Blood glucose levels

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

LFTs (AST, ALT and Alk-Phosphatase)

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