BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY:
Gastric hyperacidity and gastro duodenal ulcer is a very common global problem today. The etiopathogenesis of peptic ulcer has changed from Schwartz dictum ‘No acid (gastric juice)-No ulcer’ to ‘No mucosal damage-No ulcers’.
Peptic ulcer occurs in that part of the gastrointestinal tract which is exposed to gastric acid and pepsin, i.e.,the stomach and duodenum. The etiology of peptic ulcer is not clearly known. it results probably due to an imbalance between the aggressive (acid, pepsin and H.pylori) and the defensive (gastric mucus and bicarbonate secretion,prostaglandins,nitric oxide, innate resistant of the mucosal cells)factors.1
Hypersecretion of gastric acid is a pathological condition, which occurs due to uncontrolled secretion of hydrochloric acid from the parietal cells of the gastric mucosa through the proton pump H+ K+ ATPase. Even the normal rate of acid secretion may cause ulceration in the breached mucosa when some gastro protective factors are lost. Peptic ulcer disease is one of the most common gastrointestinal disorders, which causes a high rate of morbidity particularly for the population of non-industrialized countries. 2
Several factors are implicated in the pathogenesis of gastric ulcer including: increased acid-pepsin secretion, impaired bicarbonate neutralization, impaired mucus secretion and precipitate lesions on the mucosal layer. 3,4
In recent years, gastric ulcer has also been associated with infection of gastro intestinal mucosal tissue by Helicobacter pylori. About 70% of patients with peptic ulcer disease are infected by Helicobacter pylori and eradication of this microorganism seems to be curative for the disease.
Extracts of Nelumbo nucifera rhizome, flower, seeds, whole plant is used in treatingulcers,cough,fever,skipiles,biliousness,menorrhagia,thirst,diabetes,neurasthenia,spermatorrhoea,m
etorrhoea,insomnia,haemorrhage,haematosis.Hence the present study is proposed to evaluate alcoholic and aqueous extracts of whole plant of Nelumbo nucifera for antiulcer activity.5
We selected Nelumbo nucifera plant to investigate for its Anti-ulcer activity based on following evidences.
1. Nelumbo nucifera contains quercetin(Flavonoids) as one of the constituents wic is responsible for Anti ulcer activity.6
2.It has been reported that Nelumbo nucifera is having Anti-ulcer activity.5
3.Alkaloids are natural nitrogen containing secondary metabolites mostly derived from amino
acids of plants.These have been considerable pharmacological anti-ulcer activity .7
6.2 RIVEIW OF LITERATURE:
Ethnolic extract of rhizome of Nelumbo nucifera markedly reduced the blood sugar level in rats.8
Rhizome and seed extracts of Nelumbo nucifera showed immunomodulatory activity.9
Hydro alcoholic extract of Nelumbo nucifera seeds found to have antioxidant activity.10 Nelumbo nucifera semen extract improves memory in rats with scopolamine-induced amnesia through the induction of choline acetyltransferase expression.11 Methanolic extract of rhizomes of Nelumbo nucifera has shown psychopharmacological action in rats and mice.12 Rhizome and rhizome knot extract of edible lotus has shown antioxidative capacity (Nelumbo nuficera).13 Constituents of Nelumbo nucifera leaves found have antimalarial and antifungal activity.14
Nelumbo nucifera, known by a number of names including Indian Lotus, Sacred Lotus, Bean of India, or simply Lotus, is a plant in the Nelumbonaceae family. This plant is an aquatic perennial. Under favorable circumstances its seeds may remain viable for many years, with the oldest recorded lotus germination being from that of seeds 1300 years old recovered from a dry lakebed in northeastern China. Native to Greater india and Bangladesh. It is commonly cultivated in water gardens, the lotus is the national flower of India and Vietnam.15
Nelumbium speciosum Willd.
Nymphaea nelumbo 15
Kannada : Kamala,Tavaregadde.
English : East Indian lotus, Sacred lotus, Indian lotus, Egyptian lotus.5
Hindi : rugtoora
Malayalam : Thamara,Senthamara.
Tamil : Ambal,Thamarai. Bengali:Padma.
Telugu: Kalung, Erra thamara.
Perennial, aquatic herb.
Root: Stock stout, Cylindrical, embedded in the mud.
Leaves: Peltate, radially nerves, margins wavy, petiole long aculeate, inserted in the middle of the leaf.
Flowers: Large,solitary,handsome & fragrant,Rosy/white,Carpels numerous.Ovoid,fleshy,sunk separately in cavities of receptacles,maturing into nut like Achenes;skn hard & blackish brown when ripe.
Habitat : Common,cultivated in ponds & swamps.
Propagation: By rhizomes.
Part used: Leaves, Seeds, receptacles, filaments and plumule . leaves can be harvested in autumn, the flowers in summer, seeds are collected when fully ripe.16
Ecology and distribution:
Native to Greater India and Bangladesh. It is commonly cultivated in water gardens, the lotus is the national flower of India and Vietnam.
The rhizome contains many alkaloids. Leaves contain alkaloid nuciferine, romerine, and nor nuciferine,flavonoids:quercetin. The dried seed contains protein 17.2 %, fat 2.4 %, carbohydrate 66.6 %. Beside this it contains calcium, phosphorus, iron, ascorbic acid and sugar. The underground stem contains moisture 83.80 %, protein 2.70 %, fats 0.11%, starch 9.25 %, sucrose o.41 % and calcium. Vitamin B, vitamin C and aspirin (2 %) are also present in it.5,16
Flower- Cough, Fever, Skin eruptions, Biliousness.
Whole plant- Haemorrhage, Sterility, skin diseases, bleeding piles, menorrhagia, ulcers, thirst.
Traditional medicinal uses:
It is used in traditional herbal medicine for the treatment of ulcers, ripe seeds produces wholesome of Neurasthenia, Spermatorrhoea, metorrhoea .Leaves & seed cones in decoction are effective for insomnia, haemorrhage and haematemesis.Tthe plumules, the filaments or the receptors in the form of a decoction are used.16
6.3 OBJECTIVE OF STUDY:-
Preparation of alcoholic and aqueous extracts of whole plant of ” Nelumbo nucifera.”
To investigate preliminary phytochemical constituents of alcoholic and aqueous extracts of whole plant of ”Nelumbo nucifera”.
Determination of LD50 of the aqueous extract of “Nelumbo nucifera” as per OECD guidelines.
To establish the pharmacological profile of prepared extracts for its antiulcer activity.
MATERIAL AND METHODS:
SOURCE OF DATA:
Whole work is planned to generate data from laboratory studies i.e. experiments are performed as described in references. Experimental studies in journals and in text books available with college and various institutions.
IISc library, Bangalore.
PESCP library, Bangalore
RGUHS digital library(Helinet), Bangalore
Web sites: www.scienencedirect.com
7.1 PLAN OF WORK:
The whole study is divided in the following phases
Phase I: Collection of plant material.
The whole plant of Nelumbo nucifera will be collected from supplier and will be authentified. They will be subjected for alcoholic and aqueous extractions. The concentrated extract will be useful for our studies.
Phase II: Preparations of extracts.
Alcoholic extract and aqueous extract of whole plant of Nelumbo nucifera. Alcoholic extract is prepared by successive extraction by Maceration. The marc obtained after alcoholic extraction was macerated with water to obtain an aqueous extract.10
Phase III: Preliminary Phytochemical investigation.
Preliminary phytochemical investigation has been done in this phase as described by Khandelwal.
Phase IV: Determination of LD50 of the aqueous extract OF Nelumbo nucifera as per OECD guidelines. 17
Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs.. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose. If no mortality is observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the
previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed. 5 reversals occur in any 6consecutive animals tested. 3 consecutive animals died at one dose level.
Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term outcome (14 days).17
For determination of antiulcer activity the following experiments are to be performed.
All the models used in the pharmacological experiments will consist of the below 7 groups consisting of 6 animals in each group. Separate set of animals shall be used for individual test (or) models
Omeprazole ( Standard )
Alcoholic extract of Nelumbo nucifera
Alcoholic extract of Nelumbo nucifera
Aqueous extract of Nelumbo nucifera
Aqueous extract of Nelumbo nucifera
The dose of the extract will be selected after performing acute toxicity studies.
Evaluation of anti-ulcer activity will be done using following models
Pylorus ligation induced ulcer model
Indomethacin induced ulcer model
1.PYLORUS LIGATION INDUCED ULCER MODEL18,19
Forty two rats of either sex were randomly allotted to seven groups each consists of six animals. Group I served as the vehicle control; Group II served as the pylorus ligation control (4 h); Group III served as standard control; Group IV and V –alcoholic extract; and Group VI and
VII-aqueous extract; were administered orally respectively by gavage using feeding needle.
Albino rats of either sex weighing 150-200 g were used. After 18 hours of fasting, ulcer induction was undertaken. The rats were quickly and mildly anaesthetized with ether and the abdomen was cut open through a midline incision. The pylorus was secured and ligated with silk sutures, after which the wound was closed and the animal were allowed to recover from anesthesia.
After ligation of the pylorus, drinking water was withheld and the gastric examinations were under-taken 19 hours after pylorus ligation. The animals were sacrificed with an overdose of ether and the stomachs were removed after clamping the esophagus. The gastric contents were collected through the esophagus. The gastric juice was centrifuged and volume was recorded.10
Total acidity of gastric juice
An aliquot of 1ml of gastric juice was taken in to a 50 ml conical flask and two drops of phenolphthalein indicator was added and titrated with 0.01N NaOH until a permanent pink color was established. The volume of 0.01N NaOH consumed was noted. The total acidity was expressed as meq/l by the following formula: N × 0.01× 36.45 × 1000; which N is volume of NaOH consumed, 40.0 is molecular weight of NaOH, 0.01 is normality of NaOH and 1000 is the factor to be respected in liter.
Macroscopic evaluation of stomach
The stomachs were opened along the greater curvature, rinsed with saline to remove gastric contents and blood clots and examined by a ×5 magnifier lens to assess the formation of ulcer. The number of ulcers was counted. Ulcer scoring was undertaken.
The scores were: 0 = no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation.Ulcer area was assessed by using 3 M scaled surgical transpore tapes, which was fixed on a light and transparent sheet. Each cell on the tape was 1mm2 in area, so the number of cells was counted and the ulcer area was measured for each stomach.
Ulcer index was measured by using following formula.
UI = UN + US + UP × 10-1
which UI = ulcer index, UN = mean of ulcer number, US =mean of ulcer score, UP = ulcer probability (incidence %) for each group.13
2. INDOMETHACIN INDUCED ULCER MODEL18,20
Forty two rats of either sex were randomly allotted to seven groups each consists of six animals. Group I served as the vehicle control; Group II served as the indomethacin control; Group III served as standard control; Group IV and V –alcoholic extract; and Group VI and VII-aqueous extract; were administered orally respectively by gavage using feeding needle. Albino rats of either sex weighing 150-200 g were used. The test drugs are administered orally in 0.1% Tween 80 solution 10 min prior to oral indomethacin in a dose of 20 mg/kg (4 mg/ml dissolved in 0.1%Tween 80solution). Six hours later, the rats are sacrificed in ether anesthesia and their stomachs removed. Formol-saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greater curvature, then washed in warm water, and examined\under a 3-fold magnifier.
The scores were: 0 = no ulcer, 1= superficial ulcer, 2= deep ulcer, 3= perforation. Ulcer area was assessed by using 3 M scaled surgical transpore tapes, which was fixed on a light and transparent sheet. Each cell on the tape was 1mm2 in area, so the number of cells was counted and the ulcer area was measured for each stomach (15).Ulcer index was measured by using following Formula.
UI = UN + US + UP × 10-1
Where UI = ulcer index, UN = mean of ulcer number, US =mean of ulcer score, UP = ulcer probability (incidence %) for each group.
7.2 STATISTICAL ANALYSIS
Values will be expressed as mean ± SEM from 6 animals. Statistical difference in mean will be analyzed using one-way ANOVA (analysis of variance) following Turkey’s multiple comparison test p<0.05 will be considered statistically significant.
7.3 DOES THE STUDY REQUIRED ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS?
Yes, the entire experimental models require usage of laboratory animals.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN 7.3?
Yes ethical clearance has been obtained. (Copy enclosed)
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