In vitro Antioxidant potential of Sphaeranthus indicus and Abrus precatorius leaves extracts

 

Ashutosh Pal Jain¹*, Gajendra Pratap Choudhary²

¹Bhagyoday Tirth Pharmacy College, Sagar, Madhya Pradesh 470001, India.

²School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore, Madhya Pradesh 452001, India.

 

ABSTRACT:

Background: The purpose of this study was to evaluate the antioxidant potential of Sphaeranthus indicus and Abrus precatorius leaves. Methods: Both plant leaves powder was exhaustively extracted by soxhlet apparatus with various solvents of increasing order of polarity, petroleum ether followed by chloroform, methanol and water. The total phenol, tannins and flavonoids content were determined spectrophotometrically. Quantitative estimation of phenolic and flavonoid compounds in the extracts was estimated by using the Folin–Ciocalteu reagent and aluminum chloride colorimetric method respectively. The antioxidant potential of all these extracts was evaluated by reducing ability assay and DPPH radical scavenging assay with vitamin C as a standard. The antioxidant activity increased with dose dependence of extracts (from 20 to 200μg/ml). Results: The order for reducing power of extracts was recorded as vitamin C > methanol > water ≈ chloroform > petroleum ether and the same order was found in the DPPH assay having 50% inhibitory concentration (IC₅₀) values. The results revealed that the methanolic extract exhibited the highest percent inhibition of the reducing ability and DPPH radical scavenging assay as compared to the other extracts of both plant. Moreover, methanolic extract showed concentration dependent significant scavenging of DPPH, especially with S. indicus [IC₅₀ values 112.23±0.39 μg/ml] and A. precatorius [IC₅₀ values 101.44±0.41μg/ml], while that of the standard was 13.45±0.37μg/ml (vitamin C). Methanolic extract showed phenolics, flavonoids and tannins contents were significantly higher than all the extracts investigated in both plant. Conclusions: A correlation between the antioxidant activity and the total phenolic contents of the extracts indicated that phenolic compounds were the dominant contributors to the antioxidant activity of the plant. The results suggest that methanolic leaves extract of S. indicus and A. precatorius are source of natural antioxidants and free radical scavenging capability.

 

 

INTRODUCTION:

Free radical species viz. hydroxyl, nitric oxide (NO), superoxide work in an intricate way in the biological systems and their overproduction, deleteriously affect the membrane lipids, cellular proteins and enzymes, i.e. oxidative stress. This oxidative stress causes cell death and eventually leads to inflammatory disorders, cancer, diabetes, etc. However, antioxidants are helpful in protecting cells from such oxidative damage¹.

 

A number of artificial antioxidant, such as 2,6-di-ter-butyl-4-methylphenol(BHT),2-3-ter-butyl-4-methoxyphe nol (BHA) and ter-butylhydroquinone (TBHQ) have been added to foodstuffs but, because of their toxicity, their utilize has been troublesome. For this motive, the development and isolation of natural antioxidants from edible herbal species are in progress². Peak levels of active oxygen species or free radicals create oxidative stress, which leads to a variety of biochemical and physiological lesions and often results in metabolic impairment and cell death. Thus, the intake of natural antioxidants is very important for humans. Medicinal and edible plants are rich source of such kind of compounds³. There are favorable circumstances of plants are a renewable source of starting material in many but not all cases. If one elects to technique proposing that particular plants may yield valuable medications dependent on long term use by people (ethno medicine) can rationalize and isolated active compounds from the plants with no history of human use. It is universally believed that plants provide an unlimited source of novel and complex chemical structures and useful biologic activity, patent protection can be assured. Further, the trend today, especially in an industrial site, needs to isolate novel bioactive structures from plants, since the ultimate goal is to use the active compounds to produce synthetic derivatives with lower toxicity and higher efficacy⁴. Medicinal plants constitute an important source of active natural products which differ widely in terms of structures, biological properties and mechanisms of actions. The most effective way to eliminate free radicals which cause the oxidative stress is with the help of antioxidants. Various phytochemical components, especially polyphenols (such as flavonoids, phyenyl propanoids, phenolic acids, tannins, etc) are known to be responsible for the free radical scavenging and suppressing such disorders including atherosclerosis, ischemic heart disease, ageing, diabetes mellitus, cancer, neurodegenerative diseases and others⁵.

 

Sphaeranthus indicus Linn belongs to family Asteraceae; the most versatile medicinal plants having a wide spectrum of biological activity. It grows well in rice fields dry waste lands and also cultivated in tropical and subtropical parts of India. A wide range of phytochemical constituents have been reported from various part of S.indicus include eudesmanoids, sesquiterpenes, sphaeranthene, stigmasterol, sitosterol, geraniol, sterol and flavone glycoside. In pharma cological studies, the plant extract has shown activity against antimicrobial, antiviral, larvicidal, analgesic, antipyretic, hepatoprotective, antitussive, wound healing, bronchodilatory, mast cell stabilizing activity, anxiolytic, neuroleptic, immunomodulatory, antidiabetic, nephro protective and many other activities^6-17.

 

Abrus precatorius, locally known as Indian licorice is one of the crucial herbs commonly known as belonging to family Fabaceae, native to the Indian subcontinents. The plant leaves consist sweet-tasting unique phytoconstituent are abrusoside A to D and abrusogenin, a triterpene as well as abrus agglutinin, isoflavan quinones including abruquinones D, E and F^18,19.

 Various scientific studies reported biological activity against various diseases like anti-diarrhoeal, anti-fertility,anti-asthmatic, anti-inflammatory, anti-bacterial, anti-diabetic, anti-convulsants, immunopotentiating, antioxidant (free-radical scavengers) and anti-ulcer activities^20-27.

 

The active secondary metabolites phenolics, tannins and flavonoids contents are well-known powerful natural antioxidant; hence it is valuable to estimate their total content in the herb extract. Furthermore antioxidants may play a role in prevention of diseases such as Alzheimer’s disease, cancer, cardiovascular disease and muscular degeneration by scavenging free radicals²⁸. Last few decades natural antioxidants, as food additives for inactivating free radicals receives a lot of attention nowadays, not only for their scavenging properties, but also because they are natural, safe, and their appreciation by consumers is very favorable. The objective of the present study has been performed comparative antioxidant potential of various extracts of S. indicus and A. precatorius. Therefore, the present work investigates the possible antioxidant activity of both plant leaves to use as a natural preservative in food or functional food.

 

MATERIAL AND METHODS:

Chemicals and Reagents:

Folin-Ciocalteu reagent (Sigma-Aldrich Chemical Pvt. Ltd., India), 2, 2-Diphenyle-2-picryl hydrazyl (DPPH) is obtained from Himedia laboratories Pvt. Ltd. (Mumbai India). Vitamin C, Quercetin hydrate (95 %) and Gallic acid were obtained from Loba Chemie Pvt. Ltd. (Mumbai, India). All other chemicals used, including the solvents, were of analytical grade. All solutions, including freshly prepared distilled water. Appropriate blanks were used for individual assays.

 

Plant Materials:

The leaves of S. indicus and A. precatorius were collected from the garden of Garpahra temple Sagar, Madhya Pradesh, India, in the month August 2016 and the medicinal garden of Dr. Harisingh Gour Vishwavidyalaya, Sagar, India, in the month November 2016 respectively. The plants specimens were authenticated by botanist Dr. P. K. Khare, Professor of Botany, Dr. Harisingh Gour Vishwavidyalaya, Sagar, India. The herbarium specimen bearing voucher No. Bot/ Her/01/2017 and Bot/Her/02/2017 have been deposited in the Department of Botany, Dr. H.S. Gour Vishwavidyalaya, Sagar, India.

 

Preparation of plant extracts:

The shade-dried leaves of the plant were powdered and subjected to successive solvent extraction about 70 gram and 68 gram of S. indicus and A. precatorius leaves respectively. The leaves powder was exhaustively extracted by hot percolation method (soxhalation) with different solvents of increasing order of polarity, starting with a highly nonpolar solvents viz., petroleum ether followed by chloroform, methanol and water.

 

Estimation of total phenolics (TPC) and total tannins content (TTC):

The content of total phenolic compounds in plant various extracts of S. indicus and A. precatorius (1 ml, 1% w/v) was determined by Folin–Ciocalteu method²⁹ were mixed with Folin-Ciocalteu reagent (10 ml, previously diluted with distilled water in 1:10 ratio), vortexed and set aside for 5 mins, then Na₂CO₃ solution (10 ml, 7% w/v) was added and diluted up to 25 ml with distilled water. The absorption was read after 1.5 hr on 25± 2°C at λmax 765 nm against blank using UV-Vis spectrophotometer. The same procedure was repeated for the standard solution of gallic acid and the calibration curve was drawn. All determinations were performed in triplicate. Non-tannin phenolics were estimated by precipitating tannins of extracts with gelatin³⁰. For the estimation of non-tannin phenolics, the extract (2ml) was mixed with gelatin solution (100 mg/ml of distilled water) and allowed to stand for 15 mins at 4°C, vortexes and filtered through Whatman filter paper no. 1. The filtrate (0.5 ml) was diluted with distilled water (up to 1 ml) and non tannin phenolics were estimated by the similar method as used for TPC. TTC of both plant extracts were determined by subtracting non-tannin phenolics from total phenolics content.

 

Estimation of total flavonoids content:

The content of total flavonoids was estimated by the method of Olajuyigbe and Afolayan using quercetin as a reference compound³¹. The AlCl₃-methanolic solution (1 ml, 2% w/v) was mixed to diluted Various extracts of both plant (1ml, 1% w/v) and allowed to stand for 1 hr at 25±2°C before the absorbance was measured at λmax 420nm against blank using UV-Vis spectrophotometer. All determinations were carried out in triplicate.

 

Evaluation of antioxidant activity:

Reducing power assay:

The reducing power of both plant extracts was evaluated according to the method of Ignacimuthu (2011)³². Different concentrations of plant extracts and standard Vitamin C (Vit. C) (20- 200 μg/ml) were mixed with phosphate buffer (2.5ml, 0.2 M, pH 6.6) and 2.5 ml of 1% K₃Fe(CN)₆ and incubated at 50 ± 2°C for 20 mins. 2.5 ml of 10% Trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%), and the absorbance was measured at 700 nm. Increase in absorbance of the reaction mixture indicated reducing power.

 

DPPH radical scavenging assay:

DPPH radical scavenging activity of plant extracts were evaluated based on the method given by Milena Nikolova (2011)³³. Briefly, methanolic solution of DPPH solution (3 ml, 0.01 mM) was mixed with extracts or Vit. C (3 ml, 10-200 μg/ml) in capped test tubes and incubated at room temperature for 30 min. After incubation, absorbance of mixtures was measured at λmax 517 nm using UV-Vis spectrophotometer. The DPPH radical scavenging activity percentage was calculated by using the following formula as given below.

  A₀ – A_t

DPPH scavenging activity (%) = -------------- × 100

A₀

Where A₀ is the absorbance of the control and A_t is the absorbance of the sample.

 

IC₅₀ values denote the concentration of sample required to scavenge 50% DPPH free radicals.

 

Statistical analysis:

All results were carried out in triplicate. The content of total phenols, tannins and flavonoids was presented as mean ± SEM. The data was subjected to one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. Results were processed by the MS Excel computer programme (2007).

 

RESULTS AND DISCUSSIONS:

Estimation of phytochemical screening:

Phytochemical compositions phenolic compounds and flavonoids are the major constituents in most plants reported to possess antioxidant and effective scavenger of deleterious free radicals as well as reactive oxygen species³⁴. TPC in various extracts of both herbs was determined by measuring the intensity of blue color formed at λmax 765 nm while TTC of extracts was determined by subtracting non-tannin phenolics from the TPC. The results were expressed as compared to gallic acid equivalent (GAE) (mg of GAE/gm of extract), calculated using the regression equation y = 0.059x+ 0.006, r²= 0.999, where x is the absorbance and y is the GAE. While TFC was determined by measuring the intensity of red color formed due to reaction between flavonoids and AlCl₃ at λmax 420 nm. The TFC was expressed as compared to quercetin equivalent (mg of QE/gm of extract), calculated using the regression equation y =0.020x + 0.003, r² = 0.999, where x is the absorbance and y is the QE. The different extracts of S.indicus and A. precatorius have varied levels of TFC, TPC and TTC are shown in Table: 1. Their antioxidant activity depends on the number and location of hydroxyl groups of the flavonoid ring system and the relationship between peroxyl radical absorbing activity and the number of hydroxyl groups in flavonoids³⁵. The high quantity of phenolic and flavonoid contents of both plant leaves may contribute to its potential antioxidant property and curative ability chelating and neutralizing free radicals.

Table 1: Amount of TFC, TPC and TTC present in S. indicus and A. precatorius leaves extracts

	S. indicus			A. precatorius
Solvent used	TPC (mg GAE/gm of extract)	TFC (mg QE/gm of extract)	TTC (mg GAE/gm of extract)	TPC (mg GAE/gm of extract)	TFC (mg QE/gm of extract)	TTC (mg GAE/gm of extract)
Pet Ether	08.64 ± 0.22	03.45 ± 0.14	1.12 ± 0.10	07.56 ± 0.32	02.24 ± 0.12	1.82 ± 0.15
Chloroform	13.24 ± 0.16	18.24 ± 0.88	3.46 ± 0.08	12.28 ± 0.26	28.24 ± 0.88	4.46 ± 0.08
Methanol	54.38 ± 0.52	43.76 ± 1.08	21.45 ± 0.84	124.38 ±1.02	53.76 ± 1.08	32.45 ± 0.94
Water	25.40 ± 0.47	16.07 ± 0.45	14.84 ± 0.46	34.44 ± 0.46	06.07 ± 0.25	20.34 ± 0.46

Values are presented as mean ±SEM; (n=3). GAE, Gallic acid equivalent; QE, Quercetin equivalent.

 

Reducing ability assay:

Fig. 1 shows the reductive capabilities of the extracts compared to the standard vitamin C. High absorbance of the reaction mixture indicated reducing power of both plant extracts augmented with increasing quantity of the sample. For measurement of reductive ability, we investigated the Fe³⁺ to Fe²⁺ transformation with increasing concentration of the extracts. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity³⁶. Significant changes in reducing power were observed only after the concentration of the both plant extracts and vitamin C increased from 20 to 200 µg/mL and statistically significant. The results suggested that the reducing power was dependent on concentration. In this study, methanolic extracts of both plants leaves were found to possess higher radical-scavenging, reducing power and antioxidant activities.

 

The antioxidant activity of putative antioxidants has been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, and radical scavenging³⁷.

 

Fig.1: Reducing power of various extracts of S. indicus and A. precatorius

 

DPPH radical scavenging assay:

Determination of scavenging stable DPPH free radicals was a very fast method to evaluate the antioxidant activity of the extracts in a very short time. DPPH is a useful reagent for investigating the free radical-scavenging activities of phenolic compounds and a substrate to evaluate the antioxidative activity of antioxidants.

 

The color turns from purple to yellow the absorbance decreased when the DPPH radical was scavenged by an antioxidant through donation of hydrogen to form a stable DPPH-H molecule. The odd electron in the DPPH radical in absorbance at 517 nm responsible for the visible deep purple colour which disappeared after acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable diamagnetic molecule³⁸. The results of the DPPH scavenging activity of the extracts are as shown in Fig. 2 which showed that order of activity, methanolic extract exhibited significant comparable to those obtained from the standards.

 

The various extracts of S. indicus and A. precatorius leaves exhibited concentration dependent antioxidant activity by inhibiting DPPH radical with inhibitory concentration 50% (IC₅₀) values of 630.46 ± 0.27, 469.68 ± 0.23 μg/ml (pet ether) 314.89 ± 0.26, 246.7 ± 0.18 μg/ml (chloroform), 112.23 ± 0.39, 101.44 ± 0.41 μg/ml (methanol) and 275.5 ± 0.19, 286.51 ± 0.21 μg/ml (aqueous) respectively, while those of the standard was 13.45 ± 0.37 μg/ml (vitamin C).

 

Fig. 2: Percent inhibition by S. indicus and A. precatorius extracts in DPPH radical scavenging assay

 

Antioxidants in the various extracts of leaves reacted with DPPH consequently, the absorbencies decreased from the DPPH radical to the DPPH-H formed. The degree of yellowing indicated the scavenging potential of the extracts in terms of hydrogen donating ability. The scavenging ability of both plants was significant and corresponded to the presence of high quantity of phenolic compounds. It is, therefore, reasonable to conclude that methanolic leaves extract of S. indicus and A. precatorius are source of natural antioxidants and free radical scavenging capability by reducing ability and DPPH radical scavenging assay.

 

CONCLUSION:

This study suggested that methanolic extract of both plant leaves have become a great interest due to their possible uses as natural additives to replace synthetic ones. Today, antioxidative properties of extracts from plants might be helpful in preventing or slowing the progress of various oxidative stress-related diseases. This study was designed to investigate total phenolic compounds, flavonoids and tannins were detected in the various extracts of both plant. The results of reducing ability and DPPH radical scavenging assays showed that the extracts possess not only the antioxidant activities, but also potent free radical scavenger capability. The antioxidant activity of the extracts correlated well with the total phenolic contents and indicated that phenolic compounds are dominant contributors to the antioxidant activity of the extracts.

 

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest.

 

REFERENCES:

1.      Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. Journal of the American Oil Chemists Society. 1998; 75: 199–212.

2.      Ordonez AAL. Antioxidant activities of Sechium edule (Jacq.) Swartz extracts Food Chemistry. 2006; 97: 452–458.

3.      Ames B. Micronutrients prevent cancer and delay aging. Toxicology Letters.1998; 102: 5–18.

4.      Fabricant DS and Farnsworth N R. The value of plants used in traditional medicine for drug discovery. Environmental health perspectives. 2001; 109(1): 69-75.

5.      Maxwell SRJ. Prospects for the use of antioxidant therapies. Drugs.1995; 49(3): 345-361.

6.      Zachariah SM et al. Phytochemical and pharmacological screening of Sphaeranthus indicus Linn. for antimicrobial activity. International Journal of Pharmaceutical Sciences and Research. 2010; 1(10): 169.

7.      Vimalanathan S et al. Medicinal plants of Tamil Nadu (Southern India) are a rich source of antiviral activities. Pharmaceutical biology. 2009; 47: 422-429.

8.      Arivoli S et al. Larvicidal activity of fractions of Sphaeranthus indicus Linnaeus (Asteraceae) ethyl acetate whole plant extract against Aedes aegypti Linnaeus 1762, Anopheles stephensi Liston 1901 and Culex quinquefasciatus Say 1823 (Diptera: Culicidae)." Int. J. Mosq. Res.2016; 3: 18-30.

9.      Nanda BK et al. Analgesic and antipyretic activity of whole parts of Sphaeranthus indicus Linn. Journal of Chemical and Pharmaceutical Research.2009; 1: 207‑12.

10.   Sundari K et al. Hepatoprotective effect of ethanolic extracts of Sphaeranthus indicus (Linn.) on paracetamol‑induced liver toxicity in rats. International Journal of Applied Biology and Pharmaceutical Technology. 2011; 2: 315‑21.

11.   Nayak SS et al. Antitussive and synergistic effects of sleeping time induced by standard sedatives of Sphaeranthus indicus plant extracts. Journal of Advanced Pharmaceutical Research.2010; 1: 123‑32.

12.   Sadaf, F et al. Healing potential of cream containing extract of Sphaeranthus indicus on dermal wounds in Guinea pigs. Journal of ethnopharmacology. 2006; 107(2): 161-163.

13.   Sarpate RV et al. Bronchodilatory effect of Sphaeranthus indicus Linn. against allergen induced bronchospasm in guinea pigs. Pharmacognosy magazine. 2009; 5(19): 74.

14.   Galani VJ and Patel BG. Effect of hydroalcoholic extract of Sphaeranthus indicus against experimentally induced anxiety, depression and convulsions in rodents. International journal of Ayurveda research. 2010; 1: 87‑92.

15.   Bafna AR and Mishra SH. Protective effect of bioactive fraction of Sphaeranthus indicus Linn. against cyclophosphamide induced suppression of humoral immunity in mice. Journal of ethnopharmacology. 2006; 104(3): 426-429.

16.   Ghaisas M et al. Preventive effect of Sphaeranthus indicus during progression of glucocorticoidinduced insulin resistance in mice. Pharmaceutical biology. 2010; 48(12): 1371-1375.

17.   Mathew JE et al. Mast cell stabilizing effects of Sphaeranthus indicus. Journal of ethnopharmacology. 2009; 122(2): 394-396.

18.   Akinloye BA and Adalumo LA. Abrus precatorius leaves-a source of glycyrrhizin. Nigerian Journal of Pharmaceutical Research. 1981; 12: 405.

19.   Nwodo OF and Alumanah EO. Studies on Abrus precatorius seeds. II: antidiarrhoeal activity. Journal of ethnopharmacology. 1991; 31(3): 395-8.

20.   Ratnasooriya WD et al. Premakumara.Sperm antimotility properties of a seed extract of Abrus Precatorius. Journal of ethnopharmacology. 1991; 33(1-2): 85–90.

21.   Taur DJ et al. Antiasthmatic related properties of Abrus precatorius leaves on various models. Journal of Traditional and Complementary Medicine. 2017; 30: 1-5.

22.   Georgewill OA and Georgewill UO. Evaluation of the anti-inflammatory activity of extract of Abrus precatorious. Eastern Journal of Medicine. 2009; 14(1): 23.

23.   Zore GB et al. Activity-directed-fractionation and isolation of four antibacterial compounds from Abrus precatorius L roots. Natural product research. 2007; 21(10): 933-940.

24.   Wakawa HY et al. Effects of Aqueous Extract of Leaves of Abrus precatorius (Linn) on some Biochemical Parameters in Normal and Alloxan Induced Diabetic Rats. Asian Journal of Biochemistry. 2017; 12(2): 44-48.

25.   Adesina SK. Studies on some plants used as anticonvulsants in Amerindian and African traditional medicine. Fitoterapia. 1982; 53: 147–162.

26.   Ghosh D and Maiti TK. Immunomodulatory and anti-tumor activities of native and heat denatured Abrus agglutinin. Immunobiology. 2007; 212(7): 589-599.

27.   Palvai VR et al. Abrus precatorius Leaves: Antioxidant Activity in Food and Biological Systems, pH, and Temperature Stability. International Journal of Medicinal Chemistry. 2014; 1-7.

28.   Winterbourn CC. Nutritional antioxidants: their role in disease prevention. The New Zealand medical journal.1995; 108(1011): 447-449.

29.   Folin O and Ciocalteu V. On tyrosine and tryptophane determinations in proteins. 1927; Journal of biological chemistry. 73 (2): 627-650.

30.   Makkar HPS et al. Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. Journal of the Science of Food and Agriculture. 1993; 61: 161–165.

31.   Olajuyigbe OO and Afolayan, AJ. Phenolic content and antioxidant property of the bark extracts of Ziziphus mucronata Willd. subsp. mucronata Willd. BMC complementary and alternative medicine. 2011; 11(1): 130.

32.   Sunil C and Ignacimuthu S. In vitro and in vivo antioxidant activity of Symplocos cochinchinensis S. Moore leaves containing phenolic compounds. Food and chemical toxicology. 2011; 49(7): 1604-1609.

33.   Nikolova M.Screening of radical scavenging activity and polyphenol content of Bulgarian plant species.Pharmacognosy research. 2011; 3(4): 256.

34.   Larson RA. The antioxidants of higher plants. Phytochemistry. 1988; 27(4): 969-978.

35.   Kumar S and Pandey AK. Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal. 2013; 2013.

36.   Meir S et al. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. Journal of agricultural and food chemistry. 1995; 43(7): 1813-1819.

37.   Özen T and Kinalioğlu, K. Determination of antioxidant activity of various extracts of Parmelia saxatilis. Biologia. 2008; 63(2): 211-216.

38.   Matthäus B. Antioxidant activity of extracts obtained from residues of different oil seeds. Journal of Agricultural and Food Chemistry. 2002; 50(12): 3444-3452.

 

 

 

Received on 12.06.2020           Modified on 15.08.2020

Accepted on 05.09.2020         © RJPT All right reserved