Antioxidant activity of Hedera helix L. extracts and the main phyto constituents
Corresponding Author(s) : Khaled Nabih Zaki Rashed
International Journal of Allied Medical Sciences and Clinical Research,
Vol. 1 No. 2 (2013): 2013 Volume 1- Issue -2
The present study was carried out to evaluate antioxidant activity of Hedera helix stems extracts and also to investigate the main phytoconstituents in the bio-active extract. N-hexane, dichloromethane, ethyl acetate and methanol 80% extract were tested for free radical scavenging activity on model reaction with stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). The results showed that ethyl acetate was the most active one as antioxidant agent and phytochemical analysis of that extract revealed the presence of triterpenes, saponins, flavonoids, tannins and carbohydrates. The results suggest new chemical classes of natural antioxidant substances that could serve as selective agents for infectious diseases.
PHYTOCHEMICAL AND ACUTE TOXICITY STUDY OF LEAVES OF ARTOCARPUS HETEROPHYLLUS LAM
Chiranjib Bhattacharjee 1 Amitsankar Dutta 2
1Srikrupa Institute of Pharmaceutical Sciences, Vil. Velkatta, Kondapak (mdl), Dist. Medak, Siddipet. AP –502 277
2R.K.Pharmacy College, Kashipur. Surai – Sathiaon, Azamgarh. U. P. 276001
*Corresponding Author: Chiranjib Bhattacharjee
E -mail: firstname.lastname@example.org
Artocarpus heterophyllus Lam commonly known as jack fruit widely distributed in north east India, West Bengal and south Karnataka. In the present study intended with various phytochemical screening and toxicity studies were carried out on the leaves of Artocarpus Heterophyllus. The phytochemical study shows the presences of flavonoids, tannins, saponins and carbohydrates in methanolic and aqueous extracts. In acute toxicity study both the extract were found safe on a dose of 2000 mg/Kg.
KEYWORDS: Artocarpus heterophyllus,Phytoconsituents, Acute toxicity.
Since the tribal native practitioner of Manipur claimed that the leaves of Artocarpus heterophyllus are highly useful in many diseases and we were in search for an alternative medicine, this claim has attracted our attention and selected the plant for present study. On the basis of current literature surveyleaves of Artocarpus heterophyllus Lam, was taken for phytochemical investigation and toxicological evaluation. The phytochemicals present in a plant indicates the possible pharmacological action whereas the toxicological study indicates the possible dose to carry out the pharmacological screening models.
MATERIAL AND METHODS
Leaves of Artocarpus heterophyllus Lam were collected from the forest of Manipur. The plant was authenticated by Dr. Bisehwori Thongam (IBSD/MPHRD/M/1008). The shade-dried leaves were course powdered and this powder were packed in soxhlet column and extracted successively with petroleum ether, chloroform, methanol and aqueous. The extracts were concentrated under reduced pressure (bath temperature 50C). The dried extracts were stored in air tight container in refrigerator below 10C.
EXTRACTION WITH PETROLEUM ETHER
At first the finely ground leaves are placed in a ‘thimble’ made by a strong filter paper in a chamber of soxhlet (2000ml). The powders are extracted at 55 using round bottomed flask for 72 hrs. The extracting solvent in round bottomed flask is heated and its vapour condenses in condenser. The condensed extractant drips into the thimble containing the crude drug and extract it by contact. After completion of extraction petroleum ether is filtered and concentrated to dry mass. The extract is air dried to remove all traces of the solvent and the percentage yield was calculated. Petroleum ether extraction was done to defeating the powder.
EXTRACTION WITH CHLOROFORM
The marc left after petroleum ether extraction, is dried and subsequently extracted with 1200ml of chloroform (61) in a soxhlet using round bottomed flask for 72 hrs. Then the extract is concentrated by using rotary evaporator and dried to get the extract.
EXTRACTION WITH ETHANOL
The marc left is again packed in the soxhlet. The solvent is heated using isomentle and began to evaporate. For ethanol extraction the temperature used is 78. The extraction had for 18-20 hrs and after completion of extraction solution was evaporated to dryness under reduced pressure and controlled temperature by using rotary evaporator.
EXTRACTION WITH DISTILLED WATER
The marc left after ethanol extraction is placed in a stopper container with the distilled water (1176ml) and chloroform (24ml) and allowed to stand at room temperature for a period of 7 days with frequent agitation until the soluble matter has dissolved. Then the mixture is strained, the marc is pressed and the combined liquids are clarified by filtration. At last the solution is dried using rotary evaporator.
PRELIMINARY PHYTOCHEMICALS SCREENING
The preliminary phytochemical Screening was carried out on petroleum ether, chloroform, Ethanolic and aqueous extracts ofArtocarpus heterophyllus for qualitative identification of type of phytoconstituents present.
DETECTION OF ALKALOIDS
Extracts were dissolved individually in dilute Hydrochloric acid and filtered.
- MAYER’S TEST
- Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.
- WAGNER’S TEST
- Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.
- DRAGENDROFF’S TEST
- Filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.
Filtrates were treated with Hager’s reagent (saturated picric acid solution). Presences of alkaloids are confirmed by the formation of yellow coloured precipitate.
- DETECTION OF FLAVONOIDS
- ALKALINE REAGENT TEST
- Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.
- LEAD ACETATE TEST
- Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids.
DETECTION OF PHYTOSTEROLS
Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.
DETECTION OF TANNINS
- SALKOWSKI’S TEST
- Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes.
- LIBERMANN BURCHARD’S TEST
- GELATIN TEST
- To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins.
- About 0.5 g of the individual extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride (FeCl3) was added and observed for brownish green or a blue-black coloration.
- The alcoholic or aqueous extract is treated with lead acetate (10%) solution. White precipitate is formed.
DETECTION OF SAPONINS
Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.
0.5 gm of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.
DETECT ION OF PROTEINS AND AMINOACIDS
The extracts were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of proteins.
- XANTHOPROTEIC TEST
- NINHYDRIN TEST
To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid.
DETECTION OF CARBOHYDRATES
Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates.
- MOLISCH’S TEST
- Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates.
- BENEDICT’S TEST
- Filtrates were treated with Benedict’s reagent and heated gently. Orange red precipitate indicates the presence of reducing sugars.
Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.
Albino mice (20-30gm) of either sex were procured from Sri Venkateshwara enterprises, Bangalore. After procuring the animals were acclimatized for 10 day’s under standard husbandry conditions as follows;
Room temperature - 27 3C
Relative humidity - 65 10 %
12 hours light / dark cycle - The animals were fed with feed gold mohr, Lipton India Ltd., Bangalore and water was given ad libitum under strict hygienic conditions.This project was approved by Institutional Animal Ethical Committee (IAE/SKIPS/2011/MAY15/I/12/RATS-96/MICE-36).
The acute toxicity for methanolic and aqueous extracts of leaves of Artocarpus heterophyllus was determined in albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose (OCED Guideline No. 420) method of CPCSEA was adopted for toxicity studies.
RESULTS AND DISCUSSION
PREPARATION OF EXTRACTS
The yield, colour and consistency of extracts of leaves of Artocarpus heterophyllus, were shown in the table no 1.
Table No. 1: Colour, Consistency and Percentage yield of extracts of leaves of Artocarpus heterophyllus
|Sl. No.||Solvent||Colour and Consistency||Percentage yield|
|1||Pet. Ether||Dark Brown Sticky||5.40%|
|2||Chloroform||Dark Brown Solid||2.67%|
|3||Methanol||Very Dark Brown Sticky||7.50%|
PRELIMINARY PHYTOCHEMICAL STUDIES OF DIFFERENT EXTRACT
Preliminary phytochemical studies revealed that leavesof different extracts contain flavonoids, tannins, saponins and carbohydrates in ethanolic and aqueous extracts where as steroids are found to be present in pet. ether and extract of leaves. The results are complied in table no 2.
Table No.2: Preliminary phytochemical studies of different extract of leaves of Artocarpus heterophyllus
|Chemical constituents||Solvent Used|
The acute toxicity for methanolic and aqueous extracts of leaves of Artocarpus heterophyllus was determined in albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose (OCED Guideline No. 420) method of CPCSEA was adopted for toxicity studies. There were no sign of toxicity for first 48 hours and no animal died on 14 day of study at a dose of 2000 mg/Kg.
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