INTRODUCTION:

Medicinal herbs with biologically active compounds forms the basis for today’s modern drug. But only a small section of plants were examined scientifically for its therapeutically active compounds, but several plants have to be explored for its medicinal properties and has to be recorded. Essential oils is also a natural, highly valued pure and concentrated products used in perfume, cosmetic, food, feed, beverage as well as in pharmaceutical industries which are produced in industrialized as well as in developing countries all over the world for the development of nation’s economy.

Essential oils could act pharmacologically via blood stream, physiologically by means of effecting the systems of the body, psychologically via inhalation. The therapeutic potential of essential oils requires complete analysis. Hence, it was decided to isolate essential oil from Coriandrum sativum seed black variety using hydrodistillation process and its antioxidant, secondary metabolites, phytonutrients was studied to have an insight in to the properties of essential oil.

MATERIALS AND METHODS:

Sample collection:

The dry Coriandrum sativum seeds Black variety was purchased from a shop at Salem, Tamil Nadu, India.

Essential oil extraction:

The essential oil was extracted from 100gm of dried Coriandrum sativum seeds black variety by hydro-distillation process for 4hrs at 100^ºC. The obtained essential oil was dried over anhydrous sodium sulphate and stored at 4^ºC until tested and analyzed. The essential oil dissolved in equal volume of ethanol was used for further analysis. 0.1ml of this was used for each analysis.

SECONDARY METABOLITES:

The phenol and flavonoid content was analysed.

Determination of Total phenol content:

Total phenolic content was determined by Folil-ciocalteau method.¹ 0.1ml of sample was mixed with Folinciocalteau reagent (5 ml, 1:10 diluted with distilled water) kept for 5 min and aqueous NaCo₃ (4ml, 1M) was added. The mixture was allowed to stand for 15min and the liberated phenol was determined by colorimetric method at 765 nm. The standard curve was prepared. Total phenol values are expressed in terms of Gallic acid equivalent (mg/g of dry mass), which is a common reference compound.

Estimation of flavonoids:

The aluminium chloride method ²was used for the determination of the total flavonoid content. To the sample added 0.1ml of AlCl₃ (10%) sequentially and was shaken vigorously. Absorbance at 415 nm was recorded after 30min of incubation. A standard calibration plot was generated using known concentration of quercetin. The concentration of flavonoid in the test samples was calculated from the calibration plot and expressed as mg quercetin equivalent/g of sample.

ANTIOXIDANT ASSAYS:

Nitric oxide scavenging assay, Reducing power, Total antioxidant assay, Metal chelating activities were performed quantitatively.

Nitric oxide scavenging activity:

This procedure is based on the principle that, sodium nitroprusside in aqueous solution, at physiological pH spontaneously generates nitric oxide which interacts with oxygen to produce nitrite ions that can be estimated using Griess reagent.³ Scavengers of nitric oxide compete with oxygen, leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10mM) in phosphate buffered saline was mixed with 0.1ml of sample and incubated at room temperature for 150min. After the incubation period 0.5ml of Griess reagent was added. The absorbance of the chromophore formed was read at 546nm. Quercetin was used as positive control.

Reducing power assay:

Reducing power assay was performed.⁴ 0.1ml of sample was mixed with phosphate buffer (2.5ml, 0.2M, P^H 6.6) and potassium ferriccyanide (2.5ml %). The mixture was incubated at 50^ºc for 20min. 1.0 ml of trichloro acetic acid (10%) was added to stop the reaction, which was then centrifuged at 3000rpm for 10min. The upper layer of solution (1.5ml) was mixed with distilled water (1.5ml) and FeCl₃ (0.1ml, 0.1%) after mixing, the contents were incubated for 10 min and the absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicated increased reducing power. Vitamin C was used as positive control.

Total antioxidant capacity:

Total antioxidant capacity by phospho-molybdenum method⁵ was based on the reduction of Mo (V1) to Mo (V) by the sample analyte and the subsequent formation of green phosphate/Mo (V) complex at acidic pH. The phosphomolybdenum method is quantitative since the total antioxidant activity is expressed as number of equivalents of ascorbic acid.

Metal chelating activity:

The chelating ability of ferrous ion was estimated.⁶ Add 0.1ml of sample to a solution of 2mM FeCl₂ (0.05ml). The reaction was initiated by the addition of 5mM Ferrozine (160µl), the mixture was shaken vigorously and left standing at room temperature for 10min. Absorbance of the solution was then measured spectrophotometrically at 562nm. Standard curve was plotted using ascorbic acid. Distilled water (1.6ml) instead of sample solution was used as a control. Distilled water (160µl) instead of ferrozine was used as a blank, which is used for error correction because of unequal color of sample solution.

ANALYSIS OF PHYTONUTRIENTS:

Total carbohydrates, proteins, amino-acids were performed according to the standard prescribed methods.

Estimation of carbohydrate:

The total carbohydrate was estimated by Anthrone method.⁷ To 0.1 ml of sample added 4ml of anthrone reagent and contents were heated in a boiling water bath for 8 minutes. The tubes were cooled and read at 630nm using spectrophotometer Schimadzu Model - UV 1800. The standards were developed with glucose. Standard graph plotted was used to find out concentration of glucose present in the unknown/ sample.

Estimation of protein:

The total protein was estimated by Lowry’s method.⁸ To 0.1ml of sample added 2ml of alkaline copper reagent, mixed well and incubated for 10minutes. After the incubation period 0.2ml of Folin ciocalteau reagent (diluted in the ratio of 1: 2) was added and allowed for 30minutes incubation, then read at 660nm using spectrophotometer Shimadzu - Model UV 1800. The standards were developed with Bovine serum albumin. Standard graph plotted was used to find out concentration of protein present in unknown/ sample.

Estimation of amino acids:

The amino acid was estimated by Ninhydrin method.⁹ To 0.1 ml of sample added 1 ml of ninhydrin solution dissolved in Butanol: Acetone. Cover the test tube with a piece of paraffin film to avoid the loss of solvent due to evaporation. With gentle stirring, the reaction mixture was heated at 80-100ºC for 4-7 minutes. Cool the test tubes and the color developed was read at 570nm. Tyrosine was used for developing standards.

For all estimations readings were taken using UV spectrophotometer Schimadzu Model 1800. Standard graph was plotted for all experiments using their respective standards and the samples were plotted against the standard by taking concentration in X axis and OD in Y axis. Each experiments were performed thrice.

STASTICAL TOOL:

The Mean and Standard deviation (S) was calculated by using the following formula:

Mean = Sum of x values / n ( Number of values)

RESULTS AND DISCUSSION:

The yield of essential oil obtained was found to be 0.60 ±0.01 to 0.70±0.01ml per 100gm.

SECONDARY METABOLITES, ANTIOXIDANT ACTIVITY, PHYTONUTRIENT ANALYSIS

Table. 1. Secondary metabolites, antioxidant activities, phytonutrients

S.No

Parameters studied

Results (mg/100g)

Secondary metabolites

Total Phenolics

Total flavonoid

0.47 ±0.01

0.13 ±0.02

Antioxidant activities

Phosphomolybdenum assay

Nitric oxide scavenging assay

Reducing power activity

Metal chelating activity

0.19 ±0.01

0.28±0.08

0.27±0.03

0.10±0.02

Phytonutrient assay

Carbohydrate

Protein

Aminoacids

0.06±0.01

0.36±0.02

0.05±0.00

Values are Mean ± SD for Three experiments

The total phenolics was found to be higher (0.47±0.01mg/100g) in coriander seed essential oil compared to its flavonoid content (0.13±0.02mg/100g). Antioxidant activity was tested with different assays like phosphomolybdenum, nitric oxide scavenging, reducing power, metal chelating activity assay. Among the antioxidant activities tested, the nitric oxide scavenging activity was higher 0.28±0.08mg/100g, followed by reducing power activity 0.27±0.03mg/100g. The phosphomolybdenum activity was moderate showing 0.19±0.01mg/100g and metal chelating activity found to be lower (0.10±0.02mg/100g) in essential oil of Coriandrum sativum seeds. The nutrients such as carbohydrate, protein, amino acids were assessed. The protein content was higher according to our results showing 0.36±0.02mg/100g, whereas, the carbohydrate , amino acid content was low with the sample studied. The antioxidant activity of essential oils helps to preserve foods from the toxic effects of oxidants¹⁰ and also protects from brain dysfunction, cancer, heart disease and immune system maintainence. Antioxidant activities of essential oils might be due to the phytoconstituents present in it.¹¹Essential oil of coriander is applied in perfume preparation due to its pleasant fragrance. The secondary metabolities of plants are used as food additives, colors, insecticides, drugs etc.¹²The phytonutrients are beneficial to human health by acting as a potent antioxidant, anti-inflammatory, antiviral, antibacterial agents as well as in boosting the immune system.^13-20

CONCLUSION:

Essential oils from plants show significant antioxidant activities and are commonly used as food additive. Phenolics are organic compounds consist of hydroxyl group attached directly to a carbon atom that is a part of aromatic ring and are donated to free radicals thus preventing other compounds from oxidization. The phenolic compounds are directly related to antioxidant activity of essential oils and the amount of phenolic compound present depend upon the harvesting period of seed, its type etc. Hence, it is concluded, that essential oil of Coriandrum sativum could be applied in pharmaceutical preparations after performing complete analysis inorder to study its safety and toxicity aspects.

ACKNOWLEDGEMENT:

The author wishes her thanks to Honorable Vice-Chancellor Prof. Dr. C. Swaminathan Avl and Respected Registrar Prof. Dr. M. Manivannan Avl for their administrative support and excellent infrastructure facilities provided and also Co-ordinator, School of Bio-Sciences, Periyar University, Salem. The author would like to express her gratitude to her dedicated teachers.

REFERENCES:

1. Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Hamidinia A, Bekhradnia AR. Determination of antioxidant activity, phenol and flavonoids content of Parrotia persica Mey. Pharmacology online 2; 2008:560-567.

2. Mervat MM., Far EI, Hanan A, Taie,A. Antioxidant activities, total Anthocyanins, phenolics and flavonoids contents of some Sweet potato genotypes under stress of different concentrations of sucrose and sorbitol. Aust. J. Basic Appl. Sci., 3(4); 2009: 3609-3616.

3. Ebrahimzadeh MA, Nabavi SF, Nabavi SM. Antioxidant activities of methanol extract of Ambucus ebulus L.flower. Pak. J. Biol. Sci.,12(5); 2009d: 447-450.

4. Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agri. Food Chem.,43(1); 1995: 27-32.

5. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a Phosphomolybdenum Complex: Specific application to the determination of vitamin E. Analytical Biochemistry 269 (2); 1999: 337-341.

6. Ebrahimzadeh, M.A., Pourmorad, F., and Bekhradnia, A.R. Iron chelating activity screening, phenol and flavonoid content of some medicinal plants from Iran. Afr. J. Biotechnol., 32; 2008a: 43-49.

7. Hedge JE, Hofrelter BT. In: Carbohydrate chemistry, 17 Eds. 1962: Whistler RL, Be Miller JN, Academic press, New York.

8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol Reagent, J Biol Chem., 193; 1951: 265-275.

9. Yemm EW, Cocking EC, and Ricketts RE. The determination of amino acids with ninhydrin. Analyst., 80; 1955: 209-214.

10. Maestri DM, Nepote V, Lamarque AL, Zygadlo JA. Natural products as antioxidants.In Phytochemistry: Advances in Research; Imperato, F., Ed.; Research Signopost: Kerala, India, 2006; 105-135.

11. Abdalla AE, Roozen JP. Effect of plant extracts on the oxidative stability of sunflower oil and emulsion. Food Chem. 64(3); 1999: 323–329.

12. Zhao J.Nutraceuticals, Nutritional Therapy, Phytobnutrents and Phytotherapy for Improvements of Human Health, A Perspective on Plant Biotechnology Applications. Recent Pat on Biotechnology, 1(1); 2007: 75-97.

13. Chen JF. Plant foods and nutritional immunology: A scientific approach. Bright Ideas Press. 1996: Utah, U.S.

14. Juan CE, Maria TG, Francisco AT. Nutraceuticals: Facts and fiction. Phytochem. 68; 2007: 2986-3008.

15. Steven MD. Phytonutrients: A more natural approach towards cancer prevention. Sem.Cancer Bio. 17; 2007: 345-346.

16. Sundaram K, Chandrasekharan N. Nutritional properties of palm oil and its component. Adv. Oil Palm Res. 2; 2001:1061-1101.

17. Sundaram K. Meeting the rising health awareness: The palm oil formula. Palm Oil Dev. 43; 2005:20- 28.

18. Sundaram K, Basiron Y, Ahmad I. Market acceptance of palm oil: The nutritional angle. Palm Oil Dev. 21; 1994: 28-30.

19. Gapor A, Wan Hasamudin WH, Sulong M. Phytochemicals for nutraceuticals from the by- product of palm oil refining. Palm Oil Dev. 36; 2000:13-19.

20. Basiron Y, Sundaram K. Nutrition research on palm oil and its components: The PORIM Experience. Palm Oil Dev. 30; 1999: 35-40.

Received on 18.04.2016 Modified on 16.05.2016

Research J. Pharm. and Tech. 2016; 9(7):853-856

DOI: 10.5958/0974-360X.2016.00161.X