ENCLOSURE-I
6. Brief resume of the intended work
6.1: Need for the study
Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic inflammation in synovial tissue and joints, which leads to impaired joint function, severe pain and reduced life expectancy. This disease affects about 1% of the human population,women three times more often than men. The etiology and pathogenesis of this disease are not yet fully understood but it seems likely that an autoimmune-mediated attack on joints plays a crucial role in RA. In brief, inflammation and bone destruction may occur. Therefore, therapeutic agents developed for anti-inflammatory and immunosuppressant activity will be useful and indispensable for RA therapy. Currently, non-steroidal anti-inflammatory drugs (NSAIDs) such as Indomethacin are commonly used to treat RA disease. However, these drugs produce unwanted effects including gastric ulcer and dysfunction or the risk of cardiovascular disease. Although a panel of drugs has been developed for the treatment of RA, therapies for RA are not satisfied with these marketed drugs1.
In the Version-2 estimates for the global burden of disease 2000 study, published in the World Health Report 2002(3), RA is the 31st YLD (Years Lived with Disability) at global level, accounting for 0.8% of total global YLDs2. Onset is most frequent between the ages of 40 and 50, but people of any age can be affected3.
The management of RA was revolutionized with the advent of corticosteroids and Disease Modifying Anti-Rheumatic Drugs (DMARD’s) like Methotrexate, Sulphasalazine and Leflunomide. Conventional DMARD’s however have several limitations like slow onset of action, induction of partial remission and modest 5 years retention rates4.
Peptic ulcer occurs in the parts of the gastrointestinal tract (g.i.t.) which is exposed to gastric acid and pepsin, i.e. the stomach and duodenum. It results probably due to an imbalance between the aggressive (acid, pepsin, bile and H.pylori) and the defensive (gastric mucus and bicarbonate secretion, prostaglandins, nitric oxide, innate resistance of the mucosal cells) factors. A variety of psychosomatic, humoral and vascular derangements have been implicated and the importance of H.pylori infection as a contributor to ulcer formation and recurrence has been recognized.
In gastric ulcer, generally acid secretion is normal or low. In duodenal ulcer, acid secretion is high in half of the patients but normal in the rest, whether production of acid is normal or high, it does contribute to ulceration as an aggressive factor, reduction of which is the main approach to ulcer treatment and understanding of mechanism and control of gastricacid secretion will elucidate the targets of antisecretory drug action5.
The rich floral diversity of india has provided herbal health practitioners and other traditional healers in the country with an impressive pool of natural pharmacy from which plants are selected as ingredients to prepare herbal remedies and medicines (phyto medicine) for the treatment , management and control of variety of human ailments. Furthermore there is growing evidence that several classes of compounds found in plants exhibits anti-arthritic and anti-ulcer activity with fewer side effects. One such therapeutically useful medicinal plant of indiais C. maxima Burm[Family: Rutaceae].
The present study was planned to evaluate anti-arthritic and anti-ulcer activities of alcoholic and aqueous extracts of the leaves of C. maxima Burmin experimental animals like mice and rats as the plant was already reported to possess both anti arthritic and anti ulcer activities.
ENCLOSURE -II
6.2: Review of Literature
The review of various published Journals and Books have revealed that plant based drugs are showing promising anti-arthritic and anti-ulcer activities.
Plant Description:6
The plant C. maxima Burm is distributed through out India.
Family: Rutaceae
Synonym :7
C. decumana Watt, C. grandis (L.) Osbeck.
English : Pummelo, Shaddock.
Ayurvedic :Madhukarkatika.
Unani : Chakotra.
Siddha/Tamil : Pambalimasu.
Folk : Mahaa-nibu, Sadaaphal.
Parts used: Leaves.
Chemical constituents:
Naringin, Oil, Pinene, Linalool, Geraniol6and the root-bark contains β-sitosteroland acridone alkaloids. It alsocontains several coumarins. The alkaloidsand coumarins showedantimicrobial acitivity.The essential oil from the leavesand unripe fruits contain 20%limonin,30% nerolol, 40% nerolyl acetate and 3% geraniol.7
Medicinal uses:6
- used for painful swellings, ulcer, rheumatism, certain nervous disorders and convulsive coughs.
- Effective for diabetes.
- Relives constipation and inflamed nerves. Used also for scurvy.
- Helps in indigestion, flatulent pains, vomiting, cholera, and morbid cancer.
- Good for heart disease and coughs.
- Relives back pains.
ENCLOSURE -III
6.3 Main objectives of study
The main objective of the proposed work is to evaluate anti-arthritic and antiulcer activities of leaf extracts of C. maxima Burm in different experimental animal models, mice and rats.
In Phase -I:
- Preparation of alcoholic(AELCM) and aqueous(AQELCM)extracts with leaves of C. maxima Burm using soxhlet apparatus.
- To investigate preliminary phytochemical constituents.
- Determination of LD50 of both the extracts (AELCM, AQELCM) and dose selection as low, medium and high doses with respect to their LD50 values.
In Phase-II:
- To evaluate anti- arthritic activity of the extracts in various experimental animal modelslike
- Formaldehyde induced arthritis (paw volume)
- Collagen induced arthritis
- Adjuvant induced arthritis ( forepaws and hind paw inflammation)
- It is also planned to evaluate the following parameters in Adjuvant induced and Collagen induced arthritis.
- Physical parameters: Body weight, organ’s weight (lung, spleen, kidney, liver)
- Biochemical parameters:
Serum Glucose
Serum BUN (Blood urea nitrogen)
Serum Creatinine
Serum Total protein
Serum Albumin
Serum Cholesterol
Serum Triglyceride
Serum glutamate pyruvate transaminase ( ALT )
Serum glutamate oxaloacetate transaminase ( AST )
Serum alkaline phosphate ( ALP ).
In Phase III.
Antiulcer activity of the extracts are to be evaluated in
1. Pylorus ligation induced ulcers in rats
Parameters to be studied are:-
Ulcer index
Volume of gastric juice
Free acidity
Total acidity
PH
2. Aspirin induced ulcers in rats (ulcer index).
Parameters to be studied are:-
Ulcer index
Volume of gastric juice
Free acidity
Total acidity
PH
ENCLOSURE-IV
7. Materials and Methods
7.1 Source of the data
Whole work is planned to generate data from laboratory based experimental animal models as described in various National/International Journals and Books available with our college and other reputed Institutions of India and through e-publishing and Helinet of RGUHS, Bengaluru.
ENCLOSURE-V
7.2 Methods of collection of the data (including sampling procedure if any)
The whole study is divided into the following phases:
Phase-I:
- Preparation of different extracts [alcoholic (AELCM) and aqueous (AQELCM)] with leaves ofC. maxima Burm: Leaf powder of C. maxima Burm was extracted with alcohol. Each time before extracting with water, marc has to be dried in hot air-oven below 500c, and to be macerated with chloroform water (i.e.; chloroform acts as a preservative) for 24 h to obtain the aqueous extract. Each extract was concentrated by distilling off the solventand then evaporated to dryness on a water bath maintained at <50oc.
- Preliminary phytochemical screening:The preliminary phytochemical investigations are to be carried out with the AELCMand AQELCM for qualitative identification ofphytoconstituents by following standard procedures.
- Determination of LD50 of AELCM and AQELCM: The acute toxicity of C.maxima Burmhas to be determined by using albino mice of either sex (16-20 g) maintained under standard husbandry conditions. The animals are to be fasted for 3h prior to the experiment, and administered with single dose ofAELCM and AQELCMand observed for its mortality up to 48h study period (Short term toxicity). Based on the short-term toxicity profile, the next dose will be determined as per OECD guidelines No: 425. From the LD50 values of individual extracts. The dose of the individual extracts 1/20th,1/10 th and 1/5 th doses are to be selected and considered as low, medium and high doses respectively.8
In phase-II:
7.2:1 Determination of Anti-arthritic Activity
- Formaldehyde induced arthritis:9
Albino rats weighing between (160-200g) each group containing 6 animals are to be divided into 9 groups. The vehicle, standard/different doses of AELCM and AQELCM are to be administered for 10 days in respective groups and the details of the protocol was mentioned below.
Group 1: Normal control (vehicle treated, p.o).
Group 2: Toxicant Control (Formaldehyde 2%V/V, Intradermally)
Group 3: Standard (Diclofenac 5mg/kg i.p)
Group 4: Low dose of AELCM
Group 5: Medium dose of AELCM
Group 6: High dose of AELCM
Group 7: Low dose of AQELCM
Group 8: Medium dose of AQELCM
Group 9: High dose of AQELCM
Experimental procedure: Male albino rats weighing between (160-200g) will be divided into 9 groups of 6 rats in each. Group 1 served as normal control which given with vehicle only. Group 2 served as toxicant control, injected with 0.1ml of formaldehyde (2%V/V) into sub plantar region of hind paw and group 3 served as standard, given with Diclofenac 5mg/kg i.p . Animals in groups 4, 5,6 , 7, 8 and 9 were treated with three different doses (low, medium and high) of leafAELCM & AQELCM respectively. Groups 2,3,4,5,6 and 7,8,9 were intoxicated with single dose of 0.1 ml of formaldehyde (2%V/V).Afterwards daily the paw volume will be measured for10 days.
2. Collagen induced arthritis:10,11
Mice weighing between (16-20g) each group containing 6 animals will be divided into 9 groups.
Group 1: Normal control (vehicle treated, p.o).
Group 2: Toxicant Control (0.1ml collagen + Freund’s adjuvant, intradermally)
Group 3: Standard (Diclofenac 5mg/kg i.p)
Group 4: Low dose of AELCM
Group 5: Medium dose of AELCM
Group 6: High dose of AELCM
Group 7: Low dose of AQELCM
Group 8: Medium dose of AQELCM
Group 9: High dose of AQELCM
Experimental procedure:
Mice weighing between (16-20g) were divided into 9 groups of 6 rats in each. Group 1 served as normal control which has to be given with vehicle only. Group 2 served as toxicant control injected with 0.1ml of Collagen + Freund’s adjuvant into base of the tail intradermally and group 3 served as standard, given with diclofenac. Animals in groups 4, 5, 6 and 7, 8,9 will be treated with three different doses of extracts (low, medium and high) i.e.; AELCM and AQELCM respectively for 14 days. Purposely from day 13th to 21st, the animals are not dosed with the standard/extract. On day 21, the body weight of the all groups of animals are to be noted and the animals were anaesthetized with ether. Blood is to be collected through retro orbital puncture and serum analyzed has to be used for biochemical parameters estimation. Later the animals are to be sacrificed by overdose of ether, weight of body organs also noted.
3. Adjuvant induced arthritis:12
Albino rats weighing between (160-200g) each group containing 6 animals will be divided into 9 groups. The vehicle, standard/different doses of AELCM and AQELCM are to be administered for 21 days in respective groups and the details of the protocol was mentioned below.
Group 1: Normal control (vehicle treated, p.o).
Group 2: Toxicant Control (Freund’s adjuvant 0.1 ml, intradermally)
Group 3: Standard (Diclofenac 5mg/kg i.p )
Group 4: Low dose of AELCM
Group 5: Medium dose of AELCM
Group 6: High dose of AELCM
Group 7: Low dose of AQELCM
Group 8: Medium dose of AQELCM
Group 9: High dose of AQELCM
Experimental procedure:
Male albino rats weighing between (160-200g) are to be divided into 9 groups of 6 rats in each. Group 1 served as normal control which given with vehicle only. Group 2 served as toxicant control injected with 0.1ml of Freund’s adjuvant into sub plantar region of hind paw and group 3 served as standard, given withDiclofenac. Animals in groups 4, 5,6 and 7, 8,9 will be treated with three different doses of both extracts (low, medium and high) i.e.; AELCM and AQELCM respectively. Groups 2, 3, 4, 5,6, 7, 8 and 9 will be injected with single dose of 0.1 ml of Freund’s adjuvant and are to be treated with standard/extract for 12 consecutive days. Paw volumes of both paws are measured plethismographically and body weights are recorded on the 1st day of injection and on 21st day. On 0, 3,5,9,13,21days to study the influence of standard and extracts the volume of injected paw is to be measured again plethismographically to note the primary lesionin this phase. The severity of adjuvant induced disease is followed by measurement of non injected paw (secondary lesions) with a plethismometer. Purposely from day 13th to 21st, the animals are not dosed with the standard/extract. On 21st day, the body weight of the all groups of animals are to be noted and the animals were anaesthetized with ether. Blood is collected through retro orbital puncture for serumbiochemical analysis, later sacrificed by overdose of ether. Weight of body organs also are to be noted simultaneously.
7.2:2Determination of Anti-ulcer Activity:
1. Pylorus Ligation Model:13,14,15
Albino rats weighing between (140-200 g) will be divided into 8 groups of 6 rats in each. They will be fasted in individual cages with measures to avoid coprophgy for 24 h prior to the experiment with free access to water. Group 1 served as normal control given with vehicle only. Group 2 with standard drug, group 3, 4, 5 and 6, 7, 8 will be treated with low, medium and high doses of AELCM and AQELCM respectively. The various groups will be treated with vehicle/extract 30 min prior to pylorus ligation and the details of the protocol was given below.
Group 1: Normal animals treated with vehicle only
Group 2: Standard Ranitidine (10 mg/kg i.p)
Group 3: Low dose of AELCM
Group 4: Medium dose of AELCM
Group 5: High dose of AELCM
Group 6: Low dose of AQELCM
Group 7: Medium dose of AQELCM
Group 8: High dose of AQELCM
Experimental Procedure:
Under light ether anesthesia, the abdomen opened and the pylorus ligated and sutured. 4 h after ligation all the animals were sacrificed with excess of anesthetic ether and the stomach of each rat dissected out. Gastric juice collected into centrifuge tubes is to be centrifuged at 1000 rpm for 10 min and volume will be noted. The pH of the gastric juice will be recorded by pH meter. The gastric content is to be subjected for analysis of free and total acidity. The stomachs washed under running water focused under microscope to note the ulcers in the glandular portion. The number of ulcers per stomach scored microscopically with the help of (10x) hand-lens and scoring will be done.
2. Aspirin induced gastric ulcers:15,16
Albino rats of either sex weighing between (140-200 g) each group containing 6 animals were divided into 8 groups.
Group 1: Normal animals treated with vehicle only
Group 2: Standard Ranitidine (10 mg/kg i.p).
Group 3: Low dose of AELCM
Group 4: Medium dose of AELCM
Group 5: High dose of AELCM
Group 6: Low dose of AQELCM
Group 7: Medium dose of AQELCM
Group 8: High dose of AQELCM
Experimental Procedure:
Albino rats of either sex weighing between (140-200 g) will be divided into 8 groups of 6 rats in each. Group 1 served as normal control given with vehicle only. Group 2with standard drug, and groups 3, 4, 5 and 6, 7, 8 will be treated with low, medium and high doses of AELCM and AQELCM.After 30 min aspirin is administered at a dose of 250 mg/kg p.o, and after 6 hr rats will be sacrificed by using anesthetic ether. The stomachs are to be dissected out for determination of gastric lesions, then washed in warm water and examined for ulcers microscopically with the help of hand-lens (10x). Mean ulcer score for each animal in each group is expressed as ulcer index. Gastric juice collected into centrifuge tubes and centrifuged at 1000 rpm for 10 min and volume will be noted. The pH of the gastric juice has to be recorded by pH meter. The gastric content is subjected for analysis of free and total acidity.
STATISTICAL ANALYSIS
All results will be expressed as mean ± SEM from 6 animals. Statistical difference in mean has to be analyzed using one-way ANOVA (analysis of variance) followed by Post hoc test (Dennett’s ‘t’ test). P< 0.05* 0.01** and 0.001*** will be considered statistically significant.
ENCLOSURE –VI
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