INTRODUCTION:

Natural drugs have been a part of the evolution of human, healthcare for thousands of years. Nowadays nearly 88% of the global population turn to plant derived medicines as their first line of defense for maintaining health and compacting diseases. Plants are rich in a wide variety of secondary metabolites such as terpenoids, flavonoids, tannins, alkaloids etc. which have been found invitro to have medicinal properties. Pharmacological studies have accepted the value of medicinal plants as potential source of bioactive compounds [1]. One hundred and nineteen secondary plant metabolites derived from plants are used globally as drugs, 15% of all angiosperms have been investigated chemically and of that 74% of pharmacologically active plant derived components were discovered [2].

Phyto-chemicals from medicinal plants serve as lead compounds in antimicrobial discovery [3-5]. Nimba (Azadirachta indica) family Meliaceae commonly known as Neem is native of India and naturalized inmost of tropical and subtropical countries is of great medicinal value and distributed wide spread in the world. The Chemical constituents contain many biologically active compounds that can be extracted from Neem including alkaloids, Carotenoids, steroids flavonoids, triterpenoids, phenolic compounds, and ketones. Active constituents Azadirachtin is actually a mixture of seven isomeric compounds labeled as azadirachtin A-G and azadirachtin E is more effective [6]. Other compounds that have a biological activity are salannin, volatile oils, meliantriol and nimbin [7]. Neem leaf is effective in treating ringworm, eczema, anti-inflammatory, antihyperglycemic properties and it is used to heal chronic wounds, diabetic food and gangrene developing conditions. It is believed to remove toxins from the body, neutralize free radicals and purify the blood. It is used as anticancer agent and it has hepato-renal protective activity and hypolipidemic effects [8].

MATERIALS AND METHODS:

Collection of Plant Materials:

For present investigations samples were collected from Bagdara Ghati Chitrakoot, Satna (M.P.) in the month of March. The samples were authenticated by Dr. Manoj Tripathi, taxonomist Dept. of Pharmacognosy, Arogyadham, Deendayal Research Institute, Chitrakoot, Satna (M.P.). The voucher specimen was prepared and preserved in Govt. Autonomous Post Graduate College, Satna (M.P.) for reference. Bothe samples (leaf and stem bark) were washed under running tap water to eliminate dust and other foreign particles. Samples were dried under Tray Drier at 32⁰C.

Phyto-Chemical Investigation:

Bothe sample extracts (leaf and stem bark) were analyzed for the presence/ absence of preliminary phyto-chemicals such as terpenoids, flavonoids, saponins, alkaloids, tannins, glycosides and volatile oils by the following procedures [9-14]

Preparation of Samples for Phyto-Chemical Analysis:

2gm of each samples (leaf and stem bark) transferred in 250ml conical flask added 100 ml Ethanol for Ethanolic extract and water for water extract then kept on Rotatory flask shaker for 6 hr. left for 18 hr. for maceration procedure after maceration filtered through Whattman filter paper the filtrate was used for phytochemical analysis

Alkaloids:

A. Dragandroff’s Test:

Dissolved a few ml of alcohol or aq. Extract of drug in 5ml of distil water add 1ml HCl until an acid reaction occurs then add 1ml of Dragandriff’s reagent an orange or orange red ppt. is produced immediately.

B. Mayer’s Test:

add few drops of Mayer’s reagent to 1ml of acidic aq. Extract of the drug white or pole yellow is formed.

Carbohydrates:

A. Anthrone’s Test –to 2ml of Anthrone’s test solution add 0.5ml of aq. Extract of drug. A green or blue colour indicates the presence of Carbohydrates.

B. Fehling’s Test:- to 2ml of aq. Extract of drug add 1ml of mix. Of equal parts of Fehling’s solution ‘A’ and ‘B’ and boil the contents of the test tube for few minutes a Red or Pink-red precipitate is formed.

C. Molisch’s Test:- in a test tube containing 2ml of aq. Extract of drug add 2 drops of freshly prepared 20% alcoholic solution of α-napthol and mix pour 2ml concentrate H₂SO₄ so as to form a layer below the mixture. Carbohydrate if present Red-Violet ring, which disappear on the addition of an excess alkali solution.

Flavonoids :

In test tube containing 0.5ml of alcoholic extract of drug add 5-10 drops of dilute HCl followed by small pieces of ‘Mg’ in the presence of Flavonoids Pink, Reddish -pink or brown colour is produce

Triterpenoid’s :

Libarmann- Burchard’s Test:- add 2ml of acidic anhydrid’s solution to 1ml of petroleum ether extract of drug in chloroform followed by 1ml of concentrate H₂SO₄ a violet coloured ring is formed indicates the presence of Triterpenoids.

Proteins :

A. Bieuret’s Test:- To 1ml of hot aq. Extract of drug add 5-8 drops of 10% w/v NaOH solution followed by 1 to 2 drop of 3% w/v CuSO₄solution a Red or Violet colour is obtained.

B. Millon’s Test:- Dissolve small quantity of aq. Extract of drug in 1ml of distilled water and 5-6 drops of millon’s reagent a white precipitate is formed which turns Red on heating.

Resins:

Dissolved the water extracts in 1ml acetone and pours the solution in to 5ml distilled water turbidity indicates the presence of Resins.

Saponins:

in test tube containing about 5ml of an aq. Extract of drug add drop of solution of sodium bicarbonate (NaHCO₃) shake it vigorously and left for few minutes Honey-Comb like structure is formed

Steroids:

Liberman’s burchard’s Test; - add 2ml of acidic anhydrides solution to 1ml of petroleum ether extract of drug in chloroform followed by 1ml of concentrate H₂SO₄ Greenish colour is developed which turns to Blue.

Tannins:

To 1-2 ml of extract of water drug add few drops of 5% FeCl₃ solution a greenish colour and the presence of tennin. White Brown colour tannins.

Starch:

Dissolve 0.015g of iodine and 0.075g of KI in 5 ml of distilled water and add 2-3ml of an aq. extract of drug a blue colour is produce.

Terpenoids :

Salkowski Test:- 5ml of eatch extract was mixed in 2ml of chloroform, and concentrated H₂SO₄ (3ml) was carefully added to from a layer. A reddish brown coloration of the interface was formed to show the presence of terpinoids.

High Performance Thin Layer Chromatography (HPTLC):

For HPTLC, the powdered of leaf and stem bark 5 g of samples were extracted with 100 ml of methanol overnight, filtered and concentrated. It was applied by spotting extracted sample on pre-coated silica-gel aluminium plate 60 F₂₅₄ (5 ˟ 10 cm with 0.2 mm layer thickness Merck Germany) using Camag Linomat -5 sample applicator and a 100 μl Hamilton syringe. The samples, in the form of bands of length 6 mm, were spotted 15 mm from the bottom, 15 mm from left margin the plate and 10 mm part. Plates were developed using mobile phase consisting of Toluene: Ethyl acetate (7:3 v\v) for leaf and Toluene: Ethyl acetate (9:1v\v) for stem bark. Linear ascending development were carried out in 10 X 10cm twin through glass chamber equilibrated with mobile phase. The optimized chamber saturation time for mobile phase was 30 min. at room temperature. The length of chromatogram run was 8 cm. 20 ml of the mobile phase. Subsequent to the development, TLC plates were dried with the help of Hot Air Oven. The peak area for samples and standard were recorded with Camera photo documentation system Camag Reprostar3. Visualization of spot was made before and after derivatization (with 5% methanolic-sulphuric acid reagent) at 254 nm and 366 nm with Win cat software and R_f values noted[ (^{15- 17)}.

RESULT AND DISCUSSION:

Preliminary phytochemical analysis:

Bothe samples (leaf and stem bark) of Nimba the phytochemical tests were and results are given in (Table-1).

HPTLC finger print profile:

Bothe samples (leaf and stem bark) of Nimba High performance thin layer chromatography (HPTLC) studies of the ethanolic extract two spots of the sample extracts applied in the TLC plate. Major spots R_fvalues with colour were recorded under 366nm, after derivatization 366nm and UV light. Chromatogram profile and R_fvalues are given (Table-2 and 3 and Fig. 1-6).

Table 1: Showing Qualitative Phyto-chemical Analyses of Azadirachta indica Leaf and stem bark

S.N.	Name of tests	Observations	Result (Leaf)	Result (stem bark)
1.	Alkaloides
A	Drengdrofs	Orange colour formed	Positive	Negative
B	Wagner test	Brown colour appear	Positive	Positive
2.	Carbohydrates
A	Anthrone’s Test	Green colour appear	Positive	Positive
B	Fehhling test	Brick red ppt. formed	Positive	Positive
C	Molish test	On adding excess of alkali red violet ring disappear	Positive	Positive
3.	Flavonoide	Greenish colour formed	Negative	Positive
4.	Protein
A	Biurets test	Green colour appear	Negative	Positive
B	Millon test	Green colour appear	Negative	Positive
5.	Resin	Turbidity appear	Positive	Positive
6.	Saponine	Honey comb like structure found	Positive	Positive
7.	Tannin	Greenish colour appear	Positive	Positive
8.	Starch	Red colour appear	Negative	Positive
9..	Steroide	Light green colour formed	Positive	Positive
10.	Terpinoids	brown coloration between interface is appear	Positive	Positive
11.	Cardiac glycoside	Violet ring appear below the brown ring	Positive	Positive
12.	Anthriquinone	white colour appear	Negative	Negative

Table-2: Rf value of HPTLC finger print profile testing solution Nimba stem bark

Rf value	Before Derivatization		After Derivatization
Rf value	254nm	366nm	366nm
Rf₁	0.52(black)	0.10 (sky blue)	0.10(sky blue)
Rf₂	-	0.22(sky blue)	0.12(light yellow)
Rf₃	-	0.48 (sky blue)	0.22 (light sky blue)
Rf₄	-	0.52(florescent )	0.36 (sky blue)
Rf₅	-		0.52(yellow)

Table-3: Rf value of HPTLC finger print profile testing solution Nimba leaf

Rf value	Before Derivatization	After Derivatization
Rf value	366nm	366nm	Day light
Rf₁	0.12(red)	0.12(light red)	0.30(green)
Rf₂	0.32(red)	0.30(red)	0.32(green)
Rf₃	0.36(red)	0.32(red)	0.36(yellow)
Rf₄	0.40(red)	0.65(Sky white)	0.46(light yellow)
Rf₅	0.50(red)	0.70(red)	0.52(light yellow)
Rf₆	0.62(red)	0.92(red)	0.90(sky blue)
Rf₇	0.70 (red)	-	0.92(yellow)
Rf₈	0.88(red)	-	-
Rf₉	0.92(red)	-	-

Fig. 1-3 HPTLC fingerprint profile of Nimba stem bark

Fig. 4-6 HPTLC fingerprint profile of Nimba stem bark

ACKNOWLEDMENT:

The authors are grateful to the Organizing Secretary Deendayal Research Institute, Chitrakoot, Satna (M.P.) for providing necessary facilities.

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Received on 09.01.2016 Modified on 27.01.2016

Res. J. Pharmacognosy and Phytochem. 2016; 8(2): 65-69

DOI: 10.5958/0975-4385.2016.00012.1