New Microsoft Office PowerPoint Presentation (2)

Chapter 5

EXPERIMENTAL WORK

• Procurement of plant material:• For the present investigation Bauhinia racemosa was collected from area of mulugu, wgl

surrounds.• Identification and authentication of leaves of Bauhinia racemosa:• The plant material was taxonomically identified by the Dr.Raju, Bherampur university • Drying and size reduction:• The leaves were carefully dried in shade for 15 days. To ensure complete dryness plant roots

were kept in hot air oven at 45˚C for 5 minutes. Then roots were subjected to size reduction to make powder. The crushed mass of root was then ready for extraction.

• Preparation of extract:• • The coarse powder was extracted successively with petroleum ether, chloroform and alcohol using a

soxhlet apparatus. Finally, the aqueous extract was prepared by decoction. Each time before extracting with next solvent the powdered material will be air dried. After the effective extraction the solvent was distilled off. The extracts were dried using a rotary vacuum evaporator and stored in a desiccators until its further use.

• • Preliminary Phytochemical Analysis:• • Qualitative screening of various extracts of leaves of Bauhinia racemosa was performed for

the identification of various classes of active chemical constituents like alkaloids, carbohydrates, glycosides, proteins, amino acids, steroids etc. using different methods Raman8, Harborne9 and Wagner10. The results of preliminary phytochemical study were tabulated in Table-2. The Phytochemical study revealed the presence of steroids, flavonoids, alkaloids, coumarins, triterpenoids, tannins and carbohydrate. The preliminary phytochemical tests are helpful in finding chemical constituents in the plant material that may lead to their quantitative estimation and also in locating the source of pharmacologically active chemical compound.

Test for Alkaloids

A:Mayer’s Test: The Extract to be tested is treated with few drops of dilute 2N HCL and 0.5 ml Mayer’s reagent .White precipitate was obtained which confirm the presence of alkaloids. Wagner’s Test: The extract is treated with few

drops of 2N HCL and 0.5 ml Wagner’s reagent. Brown flocculent precipitate was obtained which confirm the

presence of alkaloids. B:Hager’s Test: The extract is treated with few drops of

dilute 2N HCL and 0.5 ml Hager’s reagent. Yellow colored precipitate was obtained which confirms the presence of

alkaloids.

Test for Carbohydrates:

• Molisch’s test: It was performed for the presence of carbohydrates. 1 ml of 10%alcoholic solution of α-napthol was added to the extract and mixed. Then 1ml of concentrated sulphuric acid was carefully poured along the sides of the test tube violet ring formed at the junction which is considered positive test for carbohydrates.

Test for steroids :

A. Salkowski reaction: to 2ml.of extract, add 2ml of chloroform and 2ml.conc.H2SO4. shake well. Chloroform layer appears red and acid layer shows greenish yellow fluorescence.

B. Liebermann-Burchard reaction: Mix 2 ml.extract with chloroform. Add 1-2ml.aceteic anhydride and 2 drops of conc.H2SO4 from the side of test tube.

Test for reducing Sugar:

A.Fehling’s test: 5ml of solution of extract was heated with equal volumes of Fehling’s solution A &

B. Transition of color from blue through green to reddish orange confirms the presence of reducing

sugarsB.Benedict’s test: 5 ml of solution of the extract was

heated with 5 ml of Benedict’s reagent .A green, yellow or orange red precipitate was considered as a

positive test for reducing sugars.

Test for Saponins

Foam’s test: A small amount of dry extract was boiled with water and allowed to cool. It was then shaken vigorously for a minute. The formation of persistent honey comb like froth

was considered as a positive test for saponins.

Test for SterolsA.Liebermann–Burchard test: A small portion of extract was

dissolved in chloroform and 2ml of Liebermann- burchard reagent was added. Appearance of bluish green was considered as positive

test for sterols and pink or violet coloration was considered as positive test for Terpenoids.

B.Salkowski test: A small portion of extract was dissolved in chloroform and treated with an equal volume of concentrated

sulphuric acid. A Red to purple color formation was considered as a positive test for Terpenoids.

C.Libermann’s reaction: mix 3ml.extract with 3ml. acetic anhydride. Heat and cool. Add few drops conc. H2SO4. Blue color appears.

Test for Tannins

• A small portion of extract was treated with 5%ferric chloride solution. Appearance of green to blue color was taken as a positive test for tannins.

• Small portion of extract was treated with lead acetate. Appearance of creamy precipitate was considered as a positive test for tannins.

Test for Proteins

Biuret test: A small portion of extract was treated with Biuret reagent.

Xanthoprotein test : Mix 3ml. T.S. with 1ml.conc. H2SO4. White ppt is formed.

Boil. Solution turns black or brownish due to Pbs formation.

Test for Amino acids :

• Ninhydrin test: Heat 3ml. extract and 3 drops 5%Ninhydrin solution in boiling water bath 10min. purple or bluish color appears.

• Test for tyrosine: heat 3ml. extract and 3 drops Million’s reagent. Solution shows dark red color.

Test for tryptophan:• To 3ml. extract and few drops of glyoxalic acid

and conc.H2SO4. Reddish violet ring appears at junction of the two layers.

s.no Chemical test Extracts

Pet.ether Chloroform Methanol Aqueous

Test for Carbohydrates

1 Molish’s test + + + +

Tests for Reducing sugars

1 Fehling’s test + + + +

2 Benedict’s test + + + +

Test for Monosaccharides

1 Barfoed’s test - - - +

Test for non-reducing polysaccharide

1 Iodine test + + + +

2 Tannic acid test for starch

+ + + +

Tests for Alkaloids

1 Mayer’s test - + - -

2 Hager’s test - + - -

3 Wagner’s test - + - -

Test for Proteins

1 Biuret Test - - - -

2 Xanthoproteic test - - - -

3 Precipitation test with -

i) Lead acetate solution5%

- - - -

ii) CuSO4 solution 5% - - - -

4 Test for proteins containing sulphur - - - -

Test for Amino acids

1 Ninhydrin test - - - -

2 Test for Tyrosine - - - -

3 Test for Tryptophan - - - -

4 Test for Cysteine - - - -

Test for Steroids

1 Salkowaski test + - + -

2 Liebermann-Burchard test

+ - + -

3 Liebermann’s reaction

+ - + -

Tests for Flavonoids

1 Shinoda test + + + +

2 Test with Lead acetateSolution

+ + + +

3 Alkaline reagent test

+ + + +

Test for Tannins and Phenolic compounds

1 Test with FeCL3 solution

+ + + -

2 Lead acetate test + + + -

3 Bromine water test + + + -

Chief chemical constituents:

Phenolics(%) 0.55 Flavonoids(%) 0.07Saponons(%) 0.62Glycosides(%) 0.54

Tannins(%) 0.09Proteins(%) 8.9

PHARMACOLOGICAL INVESTIGATION: Determination of LD50 Range:

• TOXICITY STUDIES Toxicology is the science of the adverse affects of chemical on living organisms. It is essential to evaluate the potential toxicity of a drug, which dose not exist already, as we know every drug is toxic at some level of dosage; sometimes the adverse effects are so harmful that they cause even death. The toxicity of a newly discovered drug has to be assessed in the light of purpose for which it might be used, the period which it may be administered, the effective dose and the potency and to toxicity of drug (cross land 1980).

• Determination of the dose response is crucially important since the dose response phenomenon is extremely important in toxicology, which is used to determine the minimum lethal dose of any experimental material) good man and Gillman 1996)

• Acute and chronic toxicity tests are should be carried out to determine the effect of exposing animals to a range of doses of the drug or drugs over period between one weak and even lift time of the animals, for this generally rats and dogs were used to determine whether these effects are reversible or irreversible (Laurence bennet) so toxicity testing will aim to determine the complications arising from the pharmacological action of the drugs. The following test are usually performed on laboratory animals for detection of toxicity of compound (Konkani)

• Acute toxicity test• Sub-acute toxicity tests • chronic toxicity tests• special toxicity test• Acute toxicity tests in which single dose of the drug is used in each animal on one occasion

only for the determination of LD50 or medical lethal dose (MLD) i.e. the dose which will kill 50% of the animals of a particular species. LD50 value is determined in 72hrs test procedure.

• Sub acute toxicity test in which animals are dosed daily starting at around expected therapeutic levels and increasing step wise every two or three days until toxic signs are observed. The purpose of this is to determine the maximum tolerated dose and also to indicate the natural to toxic reaction.

• Chronic toxicity tests, in which animals are closed daily for six months. Three close levels are choosen so that the high close will produce significant retardation of growth or such type of physiological changes

• Special toxicity studies, such as mutagenicity, carcinogenicity and teratogenicity . tests are to be carried out in women of child bearing age, its effect on fertility as well as its teratogenic potential are being investigated.

Determination of LD50

• This study is carried out on animals in the labotatary with a very sophisticated manner. ‘Acute toxicity test ‘determined a LD50 value of the different extracts of ………

Method:-

• The mice were divided into groups consisting of 10 mice in each group and administered different close ( ranging from 0.1 to 4gm)kg) of extracts by oral route in each case the control groups received 0.5% carboxy methyl cellulose. Orally. The animals were placed separately in open field condition for 72 hours and the no of death and signs of chemical toxicity were noted. Finally the median lethal dose or LD50 value Was derived by the method of litchfield wilcoxon (1949 ) the observed percentage mortality was converted into probit by referring to the appropriate table (kulkarni 1999) the values thus obtained were plotted against the corresponding log dose. Before plotting, the percent dose the zero and hundred were corrected (litch field wilcoxon 1949, kulkarni 1999 ) results were made fitted with straight line after regression analysis of the probit the dose corresponding to probit 5 was found to be LD50

Treatment Dose mg/kg No.of animals No.of survivals No.of death

Control(cmc ) 10 10 00

100 10 10 00

Pet.ether extract 200 10 10 00

400 10 10 00

800 10 10 00

1600 10 10 00

2000 10 10 00

2400 10 10 00

3000 10 10 00

Determination of median lethal dose of Pet.ether extract of Bauhinia racemosa

Result-----

It was observed from the table 3 that the test extract was not mortal for mice even at 3000

mg/kg dose hence, 1/10th (300mg/kg) and1/5th (400mg/kg) of this dose was selected for further

study.

Pharmacological activities:

• Animals Studies were carried out using Wistar albino rats of both

sexes weighing 180-200g. They were obtained from the Mahaveer enterprises, Hyderabad. The animals were grouped and housed in polyacrylic cages (38cm x 23cm x 10cm) with not more than six animals per cage and maintained under standard laboratory conditions (temp.25οc) with dark and light cycle (12/12h). They were allowed free access to standard dry pellet diet and water ad libitum. The rats were acclimatized to laboratory condition for 10 days before commencement of experiment.

Analgesic and anti-inflammatory activities

• Introduction: Medicinal herbs have been used as a form of therapy for the relief of pain throughout history. Natural products in general, and medicinal plants in particular, are believed to be an important source of new chemical substances with potential therapeutic efficacy. Inflammation is considered as a primary physiologic defense mechanism that helps body to protect itself against infection, burn, toxic chemicals, allergens or other noxious stimuli.

Anti-inflammatory activity

• Inflammation is a protective response to injury. It occurs in three phases,(a) oedema and swelling with accompanying pain. These results from dilatation and increased permeability of the blood vessels (veins).

• Methods :• 1. Carrageenan induced Paw Edema:• 2. Cotton pellet method.• 3. Arachidonic acid-induced hind paw edema in

rats

Analgesic• Methods

• 1. Acetic acid induced writhing• 2. Eddy’s hot plate method• 3. Tail immersion method

DISCUSSION AND RESULTS• The analgesic,antipyretic,anti-inflammatory activity were evaluated using

both chemical and thermal methods.• In present study it was found that extract does shows significant analgesic

activity in thermal. hot-plate tests are poorly affected by non-steroidal anti inflammatory drugs (NSAIDs), but they are sensitive to the analgesic effects of opioid agents.

• Centrally acting drugs such as narcotics could inhibit both early and late phases equally, the peripherally acting drugs like NSAIDs (Aspirin, oxyphenbutazone, etc..) only inhibit the late phase indicating a possible development of an inflammatory response and the release of analgesic mediators.

• The results obtained in the present study showed that and Methanol extract exert concentration or doses-dependent antinociceptive effects, The rank order of relative potencies in the tail immersion test was: ME>CH>AQ>EH. The reaction time was found to be 9.4, 8.8, 7.4, 4.2sec respectively, while the standard drug morphine was 8.6sec. The methanol extract shows most significant increased reaction time for mice than morphine.

References• 1. Ayurvedic Pharmacopeia of India. Ed I, Vol.III, V, Indian system of

Medicine & Homeopathy, Govt. of India Ministry of Health and Family Welfare. The Controller of Publication Civil Lines, Delhi, 2001: 234.

• 2. Vaidya, A.D., Devasagayam, T.P., 2007. Current Status of Herbal Drugs in India: An Overview. J Clin Biochem Nutrition 41, 1-11

• 3. Desai W.G.(1975): Aushadhisangrah (Medicinal plant in India) Shree Gajanan Book Depot Prakashan 1:pp223.

• 4. Kirtikar KR, Basu BD. Indian Medicinal Plants, Ed I, Vol. II, Bishen MAhendra Pal Singh, Dehradun, India, 1975, 842-844.

• 5. Alok, S., Shanker, C., Lalit, K.T., Mahendra, S., Rao, C.V., 2008. Herbal Medicine for Market Potential in India: An Overview. Academic Journal of Plant Sciences 1, 26-36.

• 6. Prajapati , N.D., Purohit, S.S.,Sharma, A.K; and kumar, T., “A Hand Book of Medicinal Plants”, Agrobis India , Jodhpur. 2003, p.460

• 7. Anonymous, “The wealth of India_Raw Material”, Vol. IX, CSIR, New Delhi, 1972, p.232.

• 8. Raman N. Phtochemical Technique. New Indian Publishing Agencies, New Delhi,2006: 19.