Proximate composition , phytochemical analysis and antioxidant capacity of Aloe vera , Cannabis sativa and Mentha longifolia

Plants, miracle of nature, are able to synthesize hundreds of chemical compounds for various metabolic functions. Numerous phytochemicals (secondary metabolites) with potential biological activity have been identified in most of the plant species. In order to determine the proximate composition, phytochemical analysis and antioxidant capacity of three well known selected plants species, a study was carried out during June 2016 in Khyber Pakhtunkhwa Peshawar. The plants under study were Aloe vera Linn (leaves), Cannabis sativa Linn (whole plant) and Mentha longifolia Linn (whole plant) in PCSIR Labs Complex Peshawar. The results from proximate analysis indicated that the plants contained crude protein in the range 0.447 to 0.953%, crude fiber ranged from 12.33 to 28.47 % and crude fat in the range of 5.87 to 14.86%. Furthermore, analysis showed the presences of important phytochemicals such as tannins, flavonoids, alkaloids, glycosides and saponins in the investigated species. Antioxidant activity of the selected plants by DPPH (2,2-diphenyl-1-picrylhydrazyl radical) scavenging assay and using ascorbic acid as a standard indicated that Cannabis sativa and Aloe vera has the strongest and nearly the same activity with IC50353μg/ml. These plants can be used as herbal products.


Introduction
Plants are miracle of nature and owned the most valuable medicinal properties.Medicinal plants are considered to be the backbone of traditional medicine.About 80% of the world's community lives in less developed countries and rely more on plants for their medical purposes.According to WHO, about 80% people in these countries regularly use these traditional medicines for their primary health requirements [1].About 6,000 species of higher plants have been reported in Pakistan.Out of which 12% species are known for its medicinal value [2].Literature revealed that these plants contain active chemical constituents like phytochemicals, minerals and vitamins [3].Phytochemical are chemicals produced by plants through primary or secondary metabolism.Medicinal plants having these bioactive chemicals with high proportion of antioxidants are considered fundamental in the prevention of a variety of degenerative diseases and have potential benefits to the people [4].Antioxidants are chemicals that prevent oxidation and thereby remove potentially damaging oxidizing agents from a living cell.Free radicals like reactive oxygen and reactive nitrogen species (RONS) are molecules or molecular fragments having an unpaired electron.RONS is a combined term and consists of two classes, reactive oxygen species (ROS) and reactive nitrogen species (RNS).These free radicals are considerably unstable and extremely reactive due to the presence of unpaired electrons.During the sequence of chemical reactions that produce energy for our cells in mitochondrial respiration, ROS and RNS are naturally produced as byproducts of this essential process [5].Antioxidants protect body against the harmful effects of uncontrolled reactive oxygen species and counteract their side effects.A number of radical scavenging antioxidants are common in food sources like fruits, vegetables and tea, etc were first cleaned with running tap water and then with distilled water to remove dust and sand etc.These plants materials were air dried in shade for few days.The dried plant materials were crushed to fine powder in an electric blender, stored in polythene bags and were tagged.Then different extracts were made ready by using the standard methods [23].

Preparation of extract for proximate and phytochemical analysis
Plant material (5g) was dissolved in about 90 ml water and boiled for15 minutes, cooled and was filtered by using Whatmann filter paper No. 42 and then 100 ml volume was made in volumetric flask.This solution was used in phytochemical and proximate analysis procedures.

Preparation of extracts for antioxidant analysis
Plant sample of 5g was dissolved in 100 ml of acetone and kept on orbital shaker for one week.The extract were then concentrated on rotary evaporator at 40 o C and stored for antioxidant assay determination.

Proximate analysis
Proximate analysis of the selected medicinal plants involved determination of moisture, ash, crude protein, crude fat, crude fiber, pH, total acidity and total soluble solids.All these were found out by manual methods [23].Phytochemical screening Phytochemical screening for the presence of tannins, alkaloids, flavonoids, triterpenoids, saponins, glycosides and steroids was carried out qualitatively [24,25].

Alkaloids
The presence of alkaloids was confirmed by the following tests.

Mayer's test
To aqueous extract in a test tube, small amount of Mayer's reagent was carefully added along the walls.Development of creamy precipitate indicated alkaloids in the sample.

Wagner's test
To each aqueous extract in test tube, about 1 ml of Wagner's reagent1 was added drop wise.Formation of reddish brown ppt was an indication for the presence of alkaloids.

Ammonia solution test
To the aqueous extract, little amount of 1% ammonia solution was added to the test tube.Existence of flavonoids was detected by the formation of yellow colour.

Ferric chloride test
To the extract of plant material in a test tube, a number of drops of FeCl3 solution were added, formation of green/black coloration was an indication for flavonoids.Tannins In a test tube, about 0.5g crushed plant material was heated to boiling with 20 ml of distilled water, followed by the addition of 0.1% FeCl3, formation of blue or black color indicated tannins in the sample.Saponins Powdered sample of 2g was boiled with 20 ml of distilled water for some time.Extract of 5 ml was shaken vigorously with 10 ml distilled water for 10 minutes.Persistence of froth on heating was an indication for the presence of saponins.Triterpenoids In 2 ml chloroform (CHCl3), about 5mg of the powdered sample was added, followed by the addition of 1 ml acetic anhydride very carefully along the walls and 1 ml sulphuric acid (H2SO4).Formation of reddish violet color showed the presence of triterpenoid.

Steroids
Powdered sample (1gm) was added in10 ml chloroform and concentrated H2SO4 (1 ml) was added into the tube using side walls.Two layers were formed, upper layer became red and the lower layer of H2SO4 displayed yellow color with green fluorescence and indicated steroids.Glycosides Extract of plant samples was reacted with hydrochloric acid followed by neutralization with sodium hydroxide.Then some amount of Fehling A and B was added.Formation of red precipitates indicated glycosides.

Antioxidant assay (DPPH scavenging activity)
Stable DPPH (2,2-diphenyl-1picrylhydrazyl radical) radical was used as a reagent in this method which shows absorbance at 517 nm on UV visible spectrophotometer and the presence of antioxidant in test solution decreased its concentration with time.This results in the disappearance of absorption and colour change from purple to yellow.In 95%methanol, 0.004% w/v DPPH solution was made for present research work.Ascorbic acid (vitamin C) was used as reference standard [27].DPPH scavenging (%) was calculated by following formula:

Scavenging activity (%) =
Absorbance of the control − sample absorbance absorbance of the control 100

Results and discussion
The results (

Antioxidant activity
Antioxidant activity of the acetonic extract of the selected plants was measured at various concentrations from 100 to 500µg/ml.The scavenging of free radicals (%) of plant extracted samples (

Table 1
Water is the major part of the body cells.It helps to cushion and lubricate the brain and the joints and also helps to transport nutrients and waste.
) of proximate analysis of A. vera, M. longifolia and C. sativa revealed that moisture content was found highest (6.8%) in A. vera, 5.5% C. sativa and 5.2% in M. longifolia.