Determination of Antioxidant Activity of various extracts of  Pajanelia  longifolia (Willd.)  K. Schum, isolation and characterization of Flavonoid from Ethanol extract by Column Chromatography

 

Gini E J¹*, Sivakkumar T², Kuppuswami S³

¹Chemists College of Pharmaceutical Sciences and Research, Varikoli, Kerala, India-682308

²Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu,

India – 608002

³Department of Pharmaceutics, Nirmala College of Pharmacy, Muvattupuzha, Kerala, India-686661.

 

ABSTRACT:

The present study involves the determination of antioxidant activity of aqueous and ethanolic extracts of the plant Pajanelia longifolia (Willd.) K. Schum, isolation and characterization of flavonoid from the ethanolic extract by column chromatography. DPPH assay, ABTS assay, hydroxyl radical scavenging activity, super oxide free radical scavenging activity, nitric oxide radical scavenging activity and total antioxidant activity methods were used to study the antioxidant potential of the whole plant. Among the two extracts, the alcoholic extract shows better antioxidant activity. The alcoholic extract was subjected to column chromatography by gradient elution technique and the fraction 98-102 was found to be 3',4',5,7- tetrahydroxy-3-o- rhamnoglucosyl flavonone.

 

 

 

INTRODUCTION:

The plant Pajanelia longifolia (Willd.) K. Schum is a tall deciduous tree, up to 30m height. Plant pacifies vitiated vata, kapha, inflammation, rheumatoid arthritis, sprains, edema, neuralgia, colic, diarrhoea, wounds and gouty arthritis¹. The plant was used by the folklore practitioners of Kalanjimale Range in Karnataka state for skin diseases mainly in Eczema. Various constituents like polyphenols, proteins, carbohydrates and tannins were present in the root and stem bark of  Pajanelia longifolia (Willd.) K. Schum. The root and bark powder showed significant antioxidant and antibacterial activity against Staphylococcus aureus, Eschericia coli, Lactobacillus fermentum and Bacillus species^2,3.

The bark powder extract (water, ethanol, methanol and ethyl acetate) of Pajanelia longifolia (Willd.) K. Schum revealed the presence of total polyphenols and exhibited antioxidant and antimicrobial (Vibrio parahaemolyticus and Bacillus subtilis) activities⁴.

 

Plants were used as a source of medicine for health benefits in olden days⁵. Among complementary and alternative medicines, herbal medicine is the most commonly used. Herbal treatment is the oldest system of medicine in the world with more than 2000 years of history⁶. Since the herbal medicines are more effective, cheap, easily available and moreover less toxic in nature⁷, majority of the people rely on traditional medicines for their primary health care needs⁸. The main limitation of the traditional medicine is the lack of standardization. The plants which contain phenolic and flavonoid compounds shows antioxidant activities and thus, effective in preventing diseases associated with oxidative stress⁹. 

Free radicals are the compounds generated from normal body processes and also from environmental pollutions¹⁰. They possess an unpaired electron in the outer (valance) shell of the molecule. This is the reason, why the free radicals are highly reactive¹¹ and they tend to attack the healthy cell's DNA as well as proteins and fats, causing them to deteriorate. Anti-oxidants are compounds that protect cells against the damaging effects of reactive oxygen species, such as singlet oxygen, super oxide, peroxyl radicals, hydroxyl radicals and peroxynitrite. An imbalance between antioxidants and reactive oxygen species results in oxidative stress, leading to cellular damage. Oxidative stress has linked to cancer, ageing, atherosclerosis and ischemia injury, inflammation and neurodegenerative diseases (Parkinson’s and Alzheimer’s)¹⁰.

 

Plant material and Preparation of the Extract:

Fresh plant of Pajanelia longifolia (Willd.) K. Schum belongs to the family Bignoniaceae was collected from the eastern regions of Kerala, authenticated and identified by Dr. Abraham Mathew, Assistant Professor, PG and Research Department of Botany, St. Peters College, Kolenchery, Kerala.     

 

Preliminary Phytochemical Screening- Extraction by using Soxhlet Apparatus:

The whole parts of plant was gabled for elimination of contaminants and then powdered. Air dried at room temperature and 150gm of the powdered part was successively extracted with Petroleum ether, Chloroform, Ethyl acetate, Ethanol and distilled water using Soxhlet extractor (gradient extraction). The extracts were concentrated in vacuum, weighed and properly labelled and stored in refrigerator until further use^12,13,14.

 

Determination of antioxidant activity:

DPPH Assay Method:

DPPH–(1, 1-diphenyl-2-Picryl hydrazyl):

The radical scavenging activities of different extracts were determined by using DPPH assay.  The decrease in the absorption of the DPPH solution after the addition of an antioxidant was measured at 517 nm. Ascorbic acid (10mg/ml DMSO) was used as reference. 1, 1-diphenyl-2-picryl hydrazyl is a stable free radical with red colour which turns yellow when scavenged. The DPPH assay uses this character to show free radical scavenging activity. The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written as, DPPH + [H-A] →DPPH-H + (A). Antioxidants react with DPPH and reduce it to DPPH-H and as the consequence the absorbance decreases. The degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability.  Different volumes (1.25-10µl) of plant extracts were made up to 40µl with DMSO and 2.96ml DPPH (0.1mM) solution was added. The reaction mixture incubated in dark condition at room temperature for 20 minutes. After 20 minutes, the absorbance of the mixture was read at 517nm. 3ml of DPPH was taken as control¹⁵.

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

ABTS Assay Method:

ABTS – (2, 2’- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)

 

The preformed radical monocation of 2,2’-azino-bis(3- ethyl benzothiazoline- 6- sulfonic acid)  was generated by oxidation of ABTS with potassium persulfate (a blue chromogen) and was reduced in the presence of hydrogen donating antioxidants.

 

1ml of distilled water was added to 0.2ml of various concentration of the samples and 0.16ml of ABTS solution was added and make up  to the final volume 1.36ml. Absorbance was measured after 20min at 734nm.

 

Solution I: ABTS, 20mM solution is prepared using H₂O

Solution II: Potassium persulfate, 17mM solution is prepared using H₂O

 

0.3ml of Solution II was added to 50ml of solution I. The reaction mixture was left to stand at room temperature overnight in dark before use¹⁶.

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

 

Hydroxyl radical scavenging activity method:

This assay was based on the quantification of the degradation product of 2 deoxy ribose by condensation with TBA. Hydroxyl radical was generated by the Fe³⁺ -ascorbate- EDTA -H₂O₂ system (The Fenton reaction). The reaction mixture contained in the final volume of 1 ml - 2 deoxy 2 ribose (2.8mM), FeCl₃ (100µM), EDTA (100μM), H₂O₂ (1.0mM), ascorbic acid (100μml) in KH₂PO₄-KOH buffer (20mM pH 7.4) and various concentrations (62.5 – 2000μg/ml) of the test sample. After incubation for 1hour at 37°C, 0.5 ml of the reaction mixture was added to 1ml of 2.8% TCA, then 1ml aqueous TBA was added and the mixture was incubated at 90°C for 15 minutes to develop the colour. After cooling the absorbance was measured at 532nm against a blank solution¹⁷.

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

 

Nitric oxide radical scavenging activity method:

Griess reagent:

Nitric oxide scavenging activity was measured spectrophotometrically. Sodium nitro prusside (5mmol L^-1) in phosphate buffered saline pH7.4, was mixed with different concentration of the extract (62.5-2000 μg/ml) prepared in methanol and incubated at 25°C for 30minutes. A control without the test compound, but an equivalent amount of methanol was taken. After 30minutes, 1.5ml of the incubated solution was removed and diluted with 1.5ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid and 0.1% N-1-naphthyl ethylene diaminedihydrochloride). Absorbance of the chromophore formed during diazotization of the nitrate with sulphanilamide and subsequent coupling with N-1 naphthyl ethylene diaminedihydrochloride was measured at 546nm and the percentage scavenging activity was measured with reference to the standard¹⁶.

 

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

 

Super oxide free radical scavenging activity method:

DMSO - Dimethyl sulfoxide

Superoxide is biologically important as it can form singlet oxygen and hydroxyl radical. Super oxide anion is generated in the riboflavin-NADH system by the oxidation of NADH and assayed by the reduction of NBT resulting in the formation of blue formazan product.  Different concentrations of extracts (62.5-2000μg/ml), 0.05ml of Riboflavin solution (0.12mM), 0.2ml of EDTA solution [0.1M] and 0.1ml NBT (Nitro-blue tetrazolium) solution [1.5mM] were mixed in a test tube and the reaction mixture was diluted up to 2.64ml with phosphate buffer [0.067M]. The optical density of the solution was measured at 560nm using DMSO as blank after illumination for 5min and difference in OD was determined after 30minutes incubation in fluorescent light. Absorbance was measured after illumination for 30 min. at 560 nm on UV visible spectrophotometer¹⁸.

 

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

 

Total antioxidant activity method:

0.3ml sample (extract) was obtained with 3ml of reagent solution (0.6ml H₂SO_4, 28mM sodium phosphate and 4mM ammonium molybdate). The tube containing the reaction solutions were incubated at 95⁰C for 90 minutes. The absorbance of the solution was measured at 695nm against blank after cooling to room temperature (Methanol 0.3ml) in the place of extract was used as blank. The antioxidant activity is expressed as number of gram equivalent of ascorbic acid¹⁶.

                      Control -Test

% Inhibition =------------------- X 100  

                          Control

Statistical Analysis of Data:

Experimental results for all the extracts were expressed as mean ± SD. All measurements were replicated three times. The values were calculated from non-linear regression analysis.

 

Isolation and characterization of phytoconstituents of ethanol extract of Pajanelia longifolia (Willd.) K. schum::

The ethanolic extract was subjected to column chromatography, by means of gradient elution technique¹³.

 

RESULTS AND DISCUSSION:

Determination of antioxidant activities of the selected extracts of Pajanelia longifolia (Willd.) K. Schum:

The in-vitro antioxidant activity by DPPH assay, ABTS assay, hydroxyl radical scavenging activity, super oxide radical scavenging activity, nitric oxide radical scavenging activity and total antioxidant activity were conducted as per the procedure mentioned. The antioxidant activity of plant extracts was carried at different concentrations (62.5-2000 µg/ml) to determine the IC50 (50% growth inhibition). It was found that, the percentage of growth inhibition is increasing with increasing concentration of test compounds. The results of antioxidant activities were presented in Table 1-6 and graphically represented in Figure 1-6. Results of fractionation are presented in Table 7 and spectral datas are represented in figure 7-10.

 

Table 1: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by DPPH assay method

Con(µg/ml)	%Inhibition
	Ethanol	Water
62.5	45.7449	42.4595
125	51.6199	49.3155
250	60.2213	57.6545
500	71.9256	66.6666
1000	80.8236	78.1884
2000	90.5658	90.0638
IC50	102.5	126.7

 

Table 2: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by ABTS assay method

 

Fig.1: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by DPPH assay method

 

Fig. 2: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by ABTS assay method

 

Table 3: Comparison of % inhibition of extracts of  Pajanelia longifolia (Willd.) K. Schum by hydroxyl radical scavenging activity method

Con(µg/ml)	%Inhibition
	Ethanol	Water
62.5	45.72	44.98
125	52.21	52.0085
250	64.2077	62.6784
500	72.1604	71.0389
1000	84.5687	83.3452
2000	93.3166	90.2069
IC50	97.49	101.1

 

Table 4: Comparison of % inhibition of extracts of  Pajanelia longifolia (Willd.) K. Schum by nitric oxide radical scavenging activity method

 

Fig. 3: Comparison of %inhibition of extracts of Pajanelia longifolia(Willd.) K. Schum by hydroxyl radical scavenging activity method

 

Fig. 4: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by nitric oxide radical scavenging activity method

 

Table 5: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by super oxide free radical scavenging activity method

Con(µg/ml)	%Inhibition
	Ethanol	Water
62.5	44.7350	43.9539
125	67.02	50.40
250	69.68	60.27
500	74.85	68.08
1000	76.85	72.56
2000	80.3148	80.27
IC 50	52.29	109.2

 

Table 6: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by total antioxidant activity method

 

 

Fig. 5: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schum by super free oxide free radical scavenging activity method

 

 

Fig. 6: Comparison of % inhibition of extracts of Pajanelia longifolia (Willd.) K. Schumby total antioxidant activity method

 

Table 7:  Fractionation of ethanolic extract of Pajanelia longifolia (Willd.) K. Schum by column chromatography

Fraction Number	Solvent ratio for column elution	No. of spots	Rf value
1-2	100% P.E	-Nil-	-Nil-
3-5	P.E 90%: B 10%	-Nil-	-Nil-
6-7	P.E 80%: B 20%	-Nil-	-Nil-
8-10	P.E 70%: B 30%	-Nil-	-Nil-
11-12	P.E 60%:B 40%	-Nil-	-Nil-
13-14	P.E 50%: B 50 %	One	0.8
15-17	P. E 40%: B 60 %	-Nil-	-Nil-
18-22	P.E 30%: B 70 %	-Nil-	-Nil-
23-27	P. E 20%: B 80 %	-Nil-	-Nil-
28-3	P.E 10%: B 90 %	-Nil-	-Nil-
40-43	100% B	One	0.5
44-47	B 90%:E.A 10%	One	0.9
48-52	B 80%: E.A 20%	-Nil-	-Nil-
53-57	B 70%: E.A 30%	-Nil-	-Nil-
58-61	B 60%: E.A 40%	-Nil-	-Nil-
62-66	B 50%: E.A 50%	Three	0.8,0.4,0.6
67-70	B 40%: E.A 60%	-Nil-	-Nil-
71-74	B 30%: E.A 70%	-Nil-	-Nil-
75-78	B 20%: E.A 80%	-Nil-	-Nil-
79-82	B 10%: E.A 90%	-Nil-	-Nil-
83-86	100% E.A	-Nil-	-Nil-
87-91	E. A 90%:E 10%	-Nil-	-Nil-
92-97	E.A 80%: E 20%	-Nil-	-Nil-
98-102	E.A 70%: E 30%	One	0.6
103–106	E.A 60%: E 40%	-Nil-	-Nil-
107 –111	E.A 50%: E 50%	-Nil-	-Nil-
112-118	E.A 40%: E 60%	One	0.4
119 – 123	E.A 30%: E80%	-Nil-	-Nil-
124 – 128	E.A 20%: E 70%	-Nil-	-Nil-
129 – 132	E.A 10%: E90%	-Nil-	-Nil-
133 – 136	100% E	-Nil-	-Nil-

P.E.: Petroleum ether, B: Benzene, E.A.: Ethyl acetate, E: Ethanol

 

During the column elution process, the fractions 13-14, 40-43, 44-47, 98-102 and 112-118 has a single banding pattern which was confirmed by TLC study. The compounds were subjected to spectral analysis, i.e., FTIR, MS, ¹³C NMR and ¹HNMR for structural elucidation.

 

Physical Properties and Other details of Isolated Compound:

·        Colour: Pale yellow powder

·        TLC single spot.

1     Benzene: Ethyl acetate (5:5) - R_f value 0.6

2     Chloroform: Methanol (8:2) - R_f value 0.43

 

·        Shows the presence of 3', 4' ,5, 7- tetrahydroxy-3-o- rhamnoglucosylflavonone.

 

 

Fig. 7: FT-IR Spectrum of isolated compound

 

Fig. 8: Mass spectrum of isolated compound

 

 

Fig. 9: ¹HNMR Spectrum of  isolated compound

 

 

Fig. 10: ¹³C NMR Spectrum of isolated compound

Interpretation and observation of the isolated compound:

The compound in its IR spectra exhibited absorption band in the range 3421cm^-1 indicates that the compound may contain hydroxylic group with phenolic in nature. The stretching band at 2925cm^-1 is due to the presence of aliphatic –CH stretching. A sharp and intense band at 1654cm^-1 proves the presence of aromatic ketone.

 

The bands at 1600cm^-1, 1502cm^-1 and 1457cm^-1 represents the aromatic C=C stretching vibrations. The absorption band at 1292cm^-1 is due to C-CO-C stretching. The absorption band at 1062cm^-1 is for the ether linkage (C-O-C) stretching.

 

In the mass spectrum, the m/z value of the isolated compound of the molecular ion peak is found as 609(m-1) which corresponds to the molecular formula C₂₇H₃₀O₁₆.

 

In ¹H-NMR, bands between δ6.1 – 6.8 shows aromaticity, δ7.53 – 7.55 shows presence of phenolic OH group, δ12.6 shows alkyl group having substituents (R-CH₃)_, δ5.34 -5.35 shows carbon carbon double bond, δ4.38 – 4.39 shows ester linkage, δ3.04 – 3.72 shows ether linkage and δ2.51 shows aromatic group with alkyl substituents(benzyl).

 

In ¹³C-NMR spectrum, a signal at the range δ115 -116 shows the presence of aromaticity with C=C double bonds and ketones or aldehydes. A band at range δ18-76 shows the presence of alkanes with Sp³ hybridization.

 

CONCLUSION:

The present study concludes that the aqueous and ethanolic extract of the whole plant Pajanelia longifolia (Willd.) K. Schum possess anti oxidant activity. Among these the alcoholic extract shows more antioxidant activity. So further studies were conducted for isolation and characterization of bio logically active compounds by column chromatography. A compound 3', 4', 5, 7- tetrahydroxy-3-o-rhamno glucosyl flavonone was isolated from the ethanolic extract (fraction 98-102).

 

The traditional claims as well as literatures supported that the plant were being used for the treatment of rheumatoid arthritis, wound healing, inflammation, and gouty arthritis etc. and also it contains phytochemicals like flavonoids, alkaloids, glycosides, tannins and saponins. The drugs which possess rheumatoid arthritis and wound healing activities may be antioxidant in nature. Since phenolic compounds are having good antioxidant activity, we concluded that the antioxidant activity may be due the presence of the compound 3', 4', 5, 7- tetrahydroxy-3-o-rhamnoglucosyl flavonone.

 

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Accepted on 28.10.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017; 10(10):3391-3397.