In Vitro Evaluation of Anti-Inflammatory Activity of Woodfordi Fruticosa Leaves
Yogesh Tiwari*, Bhupendra Kumar, Deepika Chauhan, Amandeep Singh
1,2Assistant Professor, Dev Bhoomi Institute of Pharmacy and Research, Dehradun, Uttarakhand, India
3 Research Scholar ,Dev Bhoomi Institute of Pharmacy and Research, Dehradun, Uttarakhand, India
4Professor, Dev Bhoomi Institute of Pharmacy and Research, Dehradun, Uttarakhand, India
ABSTRACT:
Medicinal plants contain numerous biologically active compounds which are helpful in improving the life and treatment of diseases and these are the primary source of synthetic and traditional herbal medicine.Inflammation is a body response to injury, infection or destruction characterized by heat, redness, pain, swelling and disturbed physiological function. It is triggered by the release of chemical mediators from injured tissue migrating cells.The present study was carried out by egg protein denaturation method for in-vitro assessment of anti-inflammatory activity of leaves of woodfordia fruticosa. Denaturation of tissue proteins is one of the main reasons for inflammatory diseases.
Hence the In vitro study of different extract of woodfordia fruticosa leaves showed the presence of significant anti-inflammatory activity.
KEYWORDS:Anti-inflammatory activity, Albumin,Woodfordia fruticosa.
INTRODUCTION
Since early time man has depended on nature for both health and illness. Primitive man used plants, animal parts and minerals for treating diseases. Herbal medicines are now in great demand in the developing world for primary health care not because they are inexpensive but also for better cultural acceptability, better compatibility, better use with the human body and minimum side effects. So valuable contribution to be done to develop and identify such plants which cure and prevention thedisease (Jarald and Jarald, 2007).
A World Health Organization (WHO) Expert Group defined traditional Medicines as the sum total of all knowledge and practices, whether explicable or not, used in diagnosis prevention and elimination of physical, mental, or social imbalances and relying exclusively on practical experience and observation handed down from generation, whether verbally or in writing (WHO, 1976)
Worldwide, there is several typestraditional system of medicines can be used for the treatment of diseases. They have natural products as the main ingredient of their therapeutic preparation .AYURVEDA, SIDDHA, UNANI, HOMEOPATHY SYSTEM of medicine and even the allopathy has many drugs derived from natural sources. Ayurveda which is the oldest holistic medicines system has been derived from a Sanskrit w ord „AYUS‟(life) and „VED‟(knowledge) which means the ancient “ science of life” is believed to be prevalent for last 5000 year in India. It is one of the most
noted systemsof medicine in the world. The origin of Ayurveda has been taken from the gods. The
“Tridoshic” concept is the fundamental principle in Ayurveda. Authentic information on Ayurveda has been compiled by ancient Indian medicine practitioner in forms called SAMHITA and other similar books. It is believed that five characters of medicinal herbs that are rasa, guna, virya, vipak and prabhava can be applied to treat various pathological conditions. (kokate et al,2014)
Woodfordia fruticosa belongs to a class of medicinally important plant which, when mixed with probiotics generate enhanced activity of the medicinal value in question. A method to demonstrate such function, an issue of commercial importance, has been patented. (Das et al, 2007)
1.1 INFLAMMATION
Inflammation is a body response to injury, infection or destruction characterized by heat, redness, pain, swelling and disturbed physiological function. It is triggered by the release of chemical mediators from injured tissue migrating cells. (Gram et al, 1997)
Inflammation is a defense mechanism in the body. The immune system recognizes damaged cells, irritants and pathogens, and it begins the healing process. When something harmful or irritating affects a part of our body, there is a biological response to try to remove it. The signs and symptoms of inflammation can be uncomfortable but are a show that the body is trying to heal itself.
Inflammation is part of the body‟s immune response (Waugh, 2010).
According to Medical Dictionary of Pathology “Inflammation is a localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute or wall off both the injurious agent and injured tissue”
1.2 SYMPTOMSOFINFLAMMATION Joint pain
Joint stiffness Loss of function Redness (Rubor) Swelling (Tumor) Dolar (pain) Calor (heat)
1.3 THEAGENT CAUSING INFLAMMATION MAY BE AS 1. Physicalagent like heat,cold, radiation. Mechanical trauma.
2. Chemicalagent like organic and inorganic
3. Infectiveagent like bacteria,viruses and their toxins.
4. Immunologicalagent like cell-mediated and antigen –antibody reaction.
(Waugh, 2010)
1.4 TYPES OF INFLAMMATION Acute inflammation
Acute inflammation is short term process occurring in response to tissue injury, usually appearing within minutes or hours.
It is associated with increased vascular permeability, capillary infiltration and emigration of leukocytes. It is from the increased movement of leukocytes and plasma from bloody to the site of tissue injury. This leads to inflammatory response through various biochemical events.
ChronicInflammation
Itrefers to a prolonged inflammatory response that involves a progressive change in the type of cells present at the site of inflammation. It is associated with infiltration of mononuclear immune cells, macrophages, monocytes, neutrophils, fibroblast activation, proliferation and fibrosis. (Tripathi, 2015)
This leads to a destruction of the tissue and proceeds to heal the damaged site.
1.5 TREATMENT
Professional rehabilitation therapists treat pain and inflammation. Such treatment makes movement easier and enables people to participate more fully rehabilitation.
There are many drugs available to decrease joint pain, swelling, and inflammation and prevent or minimize the progression of the inflammatory disease.
1.6 CLASSIFICATION Selective COX-2 inhibitors
Celecoxib, Etoricoxib , Parecoxib
Nonselective-COX inhibitors which are used for the treatment.
CLASS DRUG
Salicylic Aspirin
Propionic acid Ibuprofen, ketoprofen
Anthranilic acid derivative Mefenamic acid Aryl acetic acid derivative Dicofenac, Aceclofenac
Oxicam derivative Piroxicam, Tenoxicam
Pyrrolo-pyrrole derivative Ketorolac
Indole derivative Indomethacin
Pyrazolone derivatives Phenylbutazone
Preferential COX-2 inhibitor Meloxicam, Nabumetone Selective COX-2 inhibitor Celecoxib, Etoricoxib Preferential COX-2 inhibitor
Nimesulid, Diclofenac, Aceclofenac , Meloxicam etc.
Analgesic-Antipyretic with Poor Anti-inflammatory Action Para-aminophenol derivative paracetamol Pyrazolone derivative Metamizo Benzoxazocine derivative Nefopam
SOMEHERBS HAVE ANTI-INFLAMMATORY PROPERTIES-
Turmeric- (curcuma longa) – treating arthritis, Alzheimer,s disease, and some other inflammatory condition.
Cannabis – Contain a cannabinoid called cannabichromene, which has been shown to have anti- inflammatory properties.
Ginger – Used as carminative dyspepsia, constipation
Neem – (Azadirachta indica) used as leprosy, cardiovascular disease.
1.7COLLECTION AND AUTHENTICATION OF PLANT
The leaves of the plant Woodfordia Fruticosa was collected from Bhauwala, Dehradun (Uttarakhand) and was authenticated at Botanical Survey of India (BSI, Dehradun) with Acc.No, 109.
The fresh leaves were washed under running tap water to remove adhere dirt, followed by rinsing with distilled water, shade dried and use grinder to obtain coarse powder.
1.8PREPARATION OF EXTRACT
Total gram of powered drug is weigh accurately and then 10 gm of powder drug was weighed and soaked with 100ml of distilled water , then again 10 gm of drug is weighed and soaked with 100ml of ethanol, again 10 gm of drug is weighed and soaked it with 100 ml of chloroform in a sealed container. Shake it and after shaking the extraction mixture was kept for 3 days (maceration).
After 3 days the extracts are filtered through whattman filter paper. Crude extract were obtained by evaporating the solvents in a water bath at a low temp. (45-50 0c).After that the extract is kept in shade until a solid extract was obtained.
Paste from the extract obtained was used to phytochemical screening.
1.9DETERMINATIONOFEXTRACTIVEVALUE ( Trease and Evans, 2009)
Extractive values are useful for the evaluation of a crude drug. The extractive value is defined as the amount of extract present in the crude drug. The extract is basically chemical constituent which are present in the drug. The extractive value of the crude drug determines the quality as well as purity of the drug. The chemicals are of two types. They are either water soluble or Water insoluble. Some of the constituents are solvent specific; it means they dissolve in some specific solvent. Hence these also provoke a type of method known by solvent specific extraction. In this extractive value is calculated by a common method which is same for every type of extractive value depends on specific solvent. In this about 4-5 gm of powdered drug was weighed and was kept for maceration for 24 hrs with solvent. After 24 hrs the solution was filtered and the residue was discarded. To the filtrate 5 ml was discarded and to previously weighed china dish 10 ml of filtrate was taken and was evaporated under water bath. When the filtrate was distilled off by leaving a residue in dry form the china dish was cooled and then weighed to calculate the amount of extract present in filtrate.
At last percentage yield was calculated and result is given in Table No. 4.
Formula used for calculating the extractive value:-
Extractive value (%) = weight of dried extract / weight of plant material x 100
5.4 DETERMINATION OF ASH VALUE
The ash of any organic material is composed of their non-volatile inorganic components. The object of ashing crude to remove the traces of organic matter which may be interferes in an analytical determination. On incineration, the crude drugs normally produce ash which is usually consisting of carbonates, phosphates and silicates of sodium, potassium, calcium and magnesium. In certain drugs, the percentage variation of the weight of ash from sample to sample is very small and any marked difference indicates change in quality. The ash value can be determined by 3 different methods: total ash, the acid insoluble ash, the water soluble ash .
Total Ash
A high ash value indicates contamination, substitution, adulteration or carelessness in preparing crude drug for marketing. Total ash is designed to measure the total amount of material produced after complete incineration of the ground drug at high temperature as possible to remove all the carbons.
To determine total ash, weigh accurately 2-3 gm of the powdered drug in silica crucible. Incinerate the powdered drug by increasing the heat (4500 C) until the sample was free from carbon. Then cool it by keeping it indesiccators. Weigh the ash and calculate the percentage of total ash with reference to the air dried drug. Results are showed in Table No. 5.
AcidInsolubleAsh
Usedfor the determination of earthy matter present on roots, rhizomes, and also on the leaves. To
determine the acid insoluble ash value, boil the total ash obtained as above for 5 minutes with 25ml of dilute HCL.
Filter and collect the insoluble matter on the ash less filter paper, wash the filter paper with hot water , ignite in crucible , cool and kept in desiccators. Weigh the residue and calculate the acid insoluble ash of the drug. The percentage ash was calculated of acid insoluble ash with reference to the air dried drug.
WaterSolubleAsh
Water soluble ash is that part of the total ash content which is soluble in water. To the ash obtained as total ash 25 ml water was added and boiled for 5 minutes. The insoluble matter was collected on an ash less filter paper, then wash with hot water and ignited in a crucible for 15 minutes at a
temperature not exceeding 4500C. The weight of the residue was subtracted from the weight of total ash. The content of water soluble ash with reference to dried drug was calculated (Trease and Evans, 2009).
SulphatedAsh
The drug powder was taken into a crucible and was incinerated till fumes arise. Than 10 ml of conc.
Sulphuric acid was taken and incinerated matter was moistened. It was than incinerated for 15 minutes at 8000C. It was than cooled in desiccators and weighed. The procedure was repeated two times and it should be noted down that there should be noted down that there should not be deviation of 0.5 mg in two observation
5.5 DeterminationofMoistureContent
Moisture content determination is important, not only to know excess water, but also in conjunction with suitable temperature moisture will lead to the activation of enzymes and gives suitable
conditions to the proliferation of living organism.
There are various methods to determine moisture content like loss on drying, separation and measurement of moisture, chemical methods, electrometric methods and spectroscopic methods.
Moisture is an inevitable component of crude drugs, which must be eliminated as far as practicable.
The preparation of crude drug from the harvested drug plant involves cleaning to remove soil or other extraneous material followed by drying which place a very important role in the quality as well as purity of the material. Removal of dryness of the drug is important and also equally the rate at which the moisture is removed. The duration of drying process varies from a few hours to several weeks, depending upon the water content and other features of the drug. A weighed sample of crude drug is dried at 1000C and weighed periodically until more than 0.25% is lost in 1 hours drying. The total weight loss is expressed as the percentage of the initial weight of sample. The test for loss on drying determines both water and volatile matter in crude drug. It can be carried out either by heating at 1000C -1050C.
Loss on drying is the loss of mass expressed as per percent w/w. To estimate the loss on drying 5 gm of drug is accurately weighed in a dried and tared petridish. The substance is to be dried to constant
mass or for the prescribed time as specified and result is mentioned in TABLE NO. 6.
PHYTOCHEMICAL SCREENING (Khandelwal 2008,) Test for Alkaloids
Dragendroff”s Test: - 2 ml of test solution was mixed with 2 ml of Dragendroff”s reagent (Potassium bismuth iodide solution). And orange-red precipitate indicates the presence of alkaloid.
Mayer’stest: - 2 ml of test solution was mixed with 2 ml of Mayer‟s reagent (potassium mercuric iodide solution). Whitish or cream colored precipitation indicates the presence of alkaloid.
Hager’s Test:-2 ml of test solution was mixed with Hager;s reagent (saturated aqueous solution of picric acid). Yellow color precipitate indicated the presence of alkaloid.
TestforCarbohydrate
Molisch’s test (General test) :- To 2-3 ml extract was taken and few drops of alpha-napthol solution in 95% alcohol were added. Then it was shaken and conc. H2SO4 was added from sides of the test tube. Violet ring formed at the junction of two liquids.
TestforReducingSugars
Fehling’s Solution test: - 1 ml of extract was boiled on water bath with 1 ml of each of Fehling Solutions A and B. Boil for 5-10 min. First yellow, then brick red ppt. was indicates the presence of sugar.
Benedict’s test:-To 2ml of extract, 2 ml of Benedict‟s reagent was added. The mixture is heated on a boiling water bath for 5 minutes. A orange red precipitate indicates the presence of sugar.
TestforGlycosides
Foam test (saponins):- Small amount of extract was shaken with 2ml of extract. Persistence of foam produced for 10 minutes indicated the presence of saponins.
Legal test: - 3 ml of chloroform and ammonia solution (10%) was added to 2ml of extract.
Formation of pink color indicated the presence of glycoside.
Keller –killani test (cardiac glycoside) :- Treat the extract with 2 ml of glacial acetic acid , 1 drop 5% of ferric chloride solution. Add 1 ml conc. Sulphuric acid, appearance of reddish brown ring at the interface indicates the deoxysugar characteristic of Cardenolides. Appearance of violet ring below the brown ring & a greenish ring in the acetic acid layer confirmed the result.
Test for proteins
Biurettest: - Treat the test solution with few drops of 2% of copper sulphate solution , add 1ml of ethanol followed by excess of potassium hydroxide pellets. Formation of pink color in the extract layer indicates the presence of protein.
Test for proteins containing Sulphur :- 5ml of test solution was mixed with 2ml of 40% NaOH and 2 drops of 10% Lead acetate solution. Boil the mixture and solution turned black due to the formation of lead sulphide.
Test for amino acid
NinhydrinTest: - 3 ml test solution was heated with 3 drops of 5% of ninhydrin reagent on boiling water bath for 5-10 minutes. Formation of purple colour indicates the presence of amino acid.
Test for steroids
Salkowskitest:-To2 ml of extract, add 2ml of chloroform and add carefully 2ml of conc. Sulphuric acid. A red color of chloroform layer and greenish yellow color layer of acid indicates the presence of steroids.
Testforgumsandmucilage
Dilute small quantity of ethanolic extract with water, add ruthenium red solution. A pink color production shows the presence of gums and mucilage.
TestforFlavonoids
Shinodatest: - 2-3 ml of test solution was mixed with 5ml 95% ethanol then few drops of conc. HCL and 0.5 g magnesium turning were added. Pink color indicates the presence of flavonoids.
Lead acetate Test: - Treat the extract with few drops of lead acetate solution. Formation of yellow precipitate indicates the presence of flavonoids.
Test for Tannins and Phenolic compounds
1ml of test solution was mixed with 5ml of 5% FeCL3 solution. Deep blue-black color was indicates the presence of phenol.
Lead acetate Test: - Treat the extract with 3ml of 10%nlead acetate solution. A bulky white ppt indicates the presence of phenolic compound.
Take 0.5 gm the dried powdered of the drug, boil it with 20 ml of water in test tube, filter the mixture, and add few drops of 0.1% ferric chloride. Development of a brownish green or a blue – black coloration indicated the presence of tannins.
1ml of test solution was mixed with 5ml of dilute iodine solution. Transient red color gave positive result.
1ml test solution was mixed with 5ml of gelatin solution. White ppt indicates the presence of phenols and tannins.
5.6 DETERMINATIONOFIN- VITROANTI-INFLAMMATORY ACTIVITY In vitro protein denaturation by using Egg albumin
The5ml of reaction mixture was comprised of 0.2ml of eggs albumin (from fresh hen‟s egg), 2.8 ml
of phosphate buffered saline (pH 6.4) and 2ml of varying concentration of woodfordia fruticosa so that the final concentration becomes 100, 200, 300 mcg/ml. similar volume of double distilled water served as control. Then the mixture was incubated at 370C in incubator for about 15 minutes and then heated at 700C for 5 minutes. After cooling, their absorbance was measured at 660nm by using pure blank. Diclofenac sodium (standard drug) was used as reference drug and treated as such for determination of absorbance. The percentage inhibition of protein denaturation was calculated by the formula mentioned below:
Absorbance of control – Absorbance of sample
% Inhibition = --- X 100 Absorbance of control
RESULT
TABLE NO. 6.1 : Extractive value of various extracts
S. No. Typeofextract Extractive Value (%)
1. Distilled Water 12%
2. Ethanol 16%
3. Chloroform 5.2%
4. Pet. Ether 2.52%
TABLENO.6.2 : Different ash value of woodfordia fruticosa Leaves S. No. Type of ash value Observation (%)w/w
1. Total ash 5.05%
2. Acid insoluble ash 0.05%
3. Water soluble ash 0.09%
4. Sulfated ash 1.2%
TABLE NO. 6.3 : Moisturecontentof woodfordiafruticosa leaves S. No. Weight of drug
(gm)
Weight of drug with petridish
Weight of drug with petridish after drying
% yield
1. 5gm 59.31 58.91 0.67%
2. 5gm 59.31 58.84 0.79%
3. 5gm 59.31 58.80 0.86%
4. 5gm 59.31 58.75 0.95%
+Sign indicates the presence of phytochemical constituent and – sign indicates the absence of phytochemical constituents.
S. No. Test Method Ethanol Dist. Water Chloroform
1. Alkaloids Dragondroff‟s test + +
+
Mayer‟s test + + +
Hager‟s test + + +
2. Carbohydrate Molisch‟s test + + +
3. Reducing sugar Fehling‟ssolution + + -
Benedict‟s test + + -
4. Glycosides Foamtest + + +
Legaltest + + +
Killer-killanitest + +
=
5. Proteins Biurettest - - -
Protein containing Sulphur
+ + +
6. Amino acid Ninhydrin test - - -
1. Steroids Salkowski test + + +
2. Gums & mucilage Rutheniumred - - -
3. Flavonoids Shinodatest + + +
Lead acetate test + + +
4. Tannins &
phenolic compounds
5% FeCl3 Solution + + +
Lead aceatate test - - -
0.1% ferric chloride solution
+ + +
Dilute Iodine solution
+ + +
Gelatin Solution + + +
TABLE NO. 6.5 : In-Vitro anti-inflammatory activity of woodfordia fruticosa by Egg albumin protein denaturation method
S.No. Type of extract Concentration mcg/ml
Absorbance % Inhibition
1. Distilledwater 100 0.375 12.99%
2. Distilledwater 200 0.281 34.80%
3. Distilledwater 300 0.176 59.16%
4. Ethanol 100 0.134 15.18%
5. Ethanol 200 0.123 22.15%
6. Ethanol 300 0.090 43.30%
7. Chloroform 100 0.115 15.55%
8. Chloroform 200 0.110 19.11%
9. Chloroform 300 0.100 26.4%
10. Standard 50 0.227 33.43%
11. Standard 100 0.135 60.99%
12. Control/Blank - - -
Fig No. 6.1 DISCUSSION
The leaves of woodfordia fruticosawere subjected to In-Vitro anti- inflammatoryactivityinvariousconcentrationsbyegg albumin protein denaturation. The aqueous and ethanolic extract show the more anti-inflammatory activity as compare to chloroform extract.
Aqueous and alcoholic extract were found to give potent anti-inflammatory activity. The activity
might be due to the presence of high content of secondarymetaboliteswhichmay be soluble in high polarity solvents. The preliminary phytochemical investigation of woodfordiafruticosa revealed the presence of various secondary metabolites such as, carbohydrates, tannins, phenolic compounds, saponins and flavonoids, glycosides and proteins. The In-vitro anti-inflammatory activity of woodfordiafruticosa extracts was assessed by egg protein denaturation method the aqueous extract at the concentration of 300mcg/ml and the alcoholic extract at the concentration of 200mcg/ml exhibited maximum degree of anti-inflammatory activity.
SUMMARY AND CONCLUSION
TheHerbal medicines have its origin in ancient culture. Herbs have potent ingredients and should be taken with the same level of caution as pharmaceutical medication. The present study was carried out by egg protein denaturation method for in-vitro assessment of anti-inflammatory activity of leaves of woodfordia fruticosa. Denaturation of tissue proteins is one of the main reasons for inflammatory diseases. Hence the In vitro study of different extract of woodfordia fruticosa leaves showed the presence of significant anti-inflammatory activity. The aqueous extract shows more anti- inflammatory activity. The activity may be due to the presence of alkaloids, glycosides flavonoids, tannins and phenolic compounds. Protein denaturation method is important in vitro method to check anti-inflammatory activity of plant constituent. In this method Woodfordia Fruticosa revealed most significant anti-inflammatory activity.
In future aspect this plant could be beneficial in the management of inflammatory disease.
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