Inhibitory effect of Phenolic and Flavonoidal content of H. indicum Root Extract on 1,1- diphenyl-2-picrylhydrazyl radicals

 

Vivek Keshri1*, Dr. K. Rajeshwar Dutt2

1Research Scholar, Department of Pharmacy, Shri Jagdish Prasad Jhabarmal Tibrewala University,

Jhunjhun, Rajasthan, India.

2Guide, Department of Pharmacy, Shri Jagdish Prasad Jhabarmal Tibrewala University,

Jhunjhun, Rajasthan, India.

*Corresponding Author E-mail: vivekkeshri5@gmail.com

 

ABSTRACT:

The present work was targeted to determine the total phenolic and flavonoidal content along with the determination of anti-oxidant behavior of methanolic root extract of Heliotropium indicum linn. The result reveals that 400 7.31 mg Gal1ic acid equiva1ent of phenolic content and 540 59.34 mg quercetin equiva1ents of flavonoidal content was present in per gram dry weight of plant extract. The antioxidant data obtained from the work reveal that as the concentration increases the radical scavenging activity also increased. At 1000 g/ml it has shown 91% inhibition. From this study, It was understood that anti-oxidant behavior of root extract is associated with total phenolic and flavonoidal content of extract.

 

KEYWORDS: Heliotropium indicum, total phenolic content, total flavonoidal content, anti-oxidant behavior, 1,1- diphenyl-2-picrylhydrazyl (DPPH) radicals.

 

 


INTRODUCTION:

Reactive oxygen species (ROS) are the responsible cause of oxidative stress. These species are formed as by-products in the ce11s of aerobic living body. This leads to conversion of target molecules into free radicals and damage resulted1. ROS include family of oxygen-containing extremely reactive and free radicals. Examples are superoxide (O2 ), hydroxyl radicals (OH ), singlet oxygen, hydrogen peroxide (H202), and 1ipid peroxides2. ROS are having very short lives. They cause damage to main components of cell like proteins, lipid and DNA. This damage resulted in a chronic disorder like atherosclerosis, neurodegenerative disease, cancer, inflammation, aging etc3,4,5. ROS resulted in decrement of proliferation of cell and modulation of apoptosis and cell differentiation6. Epidemio1ogical evidence pointed that aging and rise in ROS are possible cause of bone loss in humans as well as in animals7.

 

Under standard physiological circumstances, cells of our body can wrestle free radical or oxidative tension by its antioxidant defense mechanism. Many endogenous guards are present inside our body, like chelating proteins, glutathione peroxidase, antioxidant enzymes cata1ase and superoxide dismutase8. Exogenous antioxidants derive from food, available in fruits and vegetab1es. Phytoconstituents like carotinoids, lycopene, polyphenols and vitamin C and E are effective antioxidant9. When endogenous or exogenous antioxidants are not able to resist oxidative damage, DNA repair enzymes, transferase, protease and lipase came into play10. When antioxidants defeated in fight against oxidative stress, disorder related to oxidative stress grow, which may include cancer, cardiovascular disease, diabetes, osteoporosis and neurological diseases11.

 

Heliotropium indicum linn. Belongs to family Boraginaceae. Hatishur is local name of H. indicum. It is an herb distributed throughout in India. In folklore systems and traditional medicine systems Heliotropium indicum was employed for treating a variety of diseases. Heliotropium indicum was used by traditional healers of Kancheepuram in Tamil Nadu for nervous disorders, skin diseases, stomachache and poison bites. In African countries, it is employed for treating malaria. Jamaican people use flower infusion for treating menorrhagia in female. In Thailand, its inflorescence is taken along with water or milk in the first three days menes for proper flow. In Eastern Nicaragua whooping cough of child is treated with leaf and root decoction12. In Cachar district of Assam juice of root is employed as opthalmia and extract of leaf is employed for treatment of wound and cuts13. People of Rayal seema of Andhra use paste of leaf for rheumatism. In Coimbatore district juice of its leaf along with coconut oil was employed to treat dandruff14.

 

Flavonoids are coming under poly-phenolic compound. In nature, flavonoids are ubiquitous. Presence of minimum 1 aromatic ring and 1 or more OH group are characteristic of phenolic compound15. These days phenols and flavonoids have received great concern due to their valuable outcomes on human. There are a small number of studies on effect of Heliotropium Indicum on free radicals by DPPH method and its total flavonoidal and phenolic contents. Thus, in present work an effort has been given to study the TFC and TPC and to estimate the antioxidant actions of methanolic Heliotropium Indicum root extract by adopting broadly accepted model of free radical scavenging system (DPPH).

 

MATERIAL AND METHODS:

The roots of H. Indicum were gather from a village of Tirunelveli district of Tamil Nadu. It was confirmed by research officer of CCRAS Mr. V. Chelladurai. It was cleansed with water and dried at normal temperature. It was then converted into fine powder by mechanical grinder. By applying Soxhalet apparatus and methanol the extract was prepared. The extract was dried by rota evaporator. The dried extract was kept at 4oC for study.

 

Total phenolic content:-

It was determined in the extract by using Folin Ciocalteau technique16. Stock so1ution of Standard Gallic acid: l0mg of ga11ic acid was disso1ved in l0 ml of distilled water to attain the concentration 1 mg/m1. Working so1ution: 50, 100, 150, 200 and 250 1 of standard gal1ic acid were obtained from stock so1ution and the vo1ume prepared up to 1 ml by addition of disti1led water to attained the fina1 concentration of 50 - 250g/m1. 1 mg/m1 of test plants extracts was arranged. The vo1ume was made to 45 m1 with disti11ed water in volumetric f1ask. l ml of Fo1in-Ciocalteu reagent (di1uted l:2 with disti1led water) was added and the content was mixed proper1y. Three minutes later, 3 m1 of 2O % sodium carbonate added to the combination and it was permitted to position for 2 hours with infrequent shaking. The absorbance of obtained blue colour that deve1oped was studied at 760 nm by using spectrophotometer.

 

Values of total phenol can be represented by Gallic acid equivalent (mg/ g of dry weight). Calculation of total phenolic content by following formula:

CV

T =---------

M

 

T=Total Phenolic amount

C= Concentration of gal1ic acid from ca1iberation curve (g/m1)

V= Volume of extract (m1)

M= Wt. of plant extract

 

Total flavonoids content:-

It was determined by employing AlCl3 colorimetric technique17. Standard Stock so1ution of Quercetin: quercetin hydrate l0mg was disso1ved in l0 ml distil1ed water to attain a concentration of l mg/ml. working solution of different concentration was prepared. The different concentation were 100 200, 300, 400 and 500ml of standard Quercetin so1ution taken from the prepared stock solution. The vo1ume made to l ml by adding distilled water to attain fina1 concentration of 100 -500 g/ml. 1mg/m1 of the plant extract was prepared. 2ml of disti1led water added to l ml extracts and mixed well. 5 minutes later, sodium nitrite (3m1, 5%) and aluminium ch1oride (0.3m1, 10%) were added. It was made to stand around 6 minutes. After that, sodium hydroxide (2 m1, 1 M) addition was made to the so1ution and vo1ume was made equal to 10 ml with addition of distilled water. A complex of red co1oured developed, which was calculated at 510 nm by using spectrophotometer.

 

Total flavonoida1 content in extracts was represented in mil1igram of quercetin equiva1ents/gdw and is calculated by the formu1a:

CV

T =---------

M

 

T=Total flavonoid amount

C= Concentration of quercetin from ca1iberation curve (g/m1)

V= Volume of extract (m1)

M= Wt. of plant extract

 

DPPH radica1 scavenging assay:-

By using DPPH (1, 1, diphenyl-2- picryhydrazyl) a free radica1, antioxidant property were calculated. Experiments were begin with preparation of DPPH (0.25mM) solution and p1ant extracts (l mg/ml, stock) so1ution in methanol. An identical extract volume was added to methanolic solutions of DPPH at different concentrations. Addition of an equal quantity of alcoho1 was done to control. The arrangement was gone under dark at room temperature and absorption was observed 20 minutes later. Ascorbic acid employed as control. Experiment was carried out in triad18.

 

A control reaction was performed devoid of test. By using blank so1ution Absorbance data were adjusted for radica1s decomposition. The inhibition by DPPH was computed by using formula given below:

% Inhibition = [1 - (Abssample / Abscontrol )] 100

 

Graph was plotted between percentage inhibition and concentration and value of IC 50 were determined. IC 50 represents antioxidant property of every sample and was represented by g/ml and was computed from the obtained inhibition curve.

 

RESULT AND DISCUSSION:

 

Graph 1- Graph of Standard (Gallic Acid) for Total phenolic content

 

Table1. Total phenol content in Gallic acid equivalent (mg/g) of H. indicum

S g/ml

D.E.W

(g)

A

GAE conc.

C g/ml

GAE conc.

C mg/ml

TPC

mg/ g

M

SEM

1000

0.001

0.878

410.5

0.410

410

400

1000

0.001

0.829

386

0.386

386

1000

0.001

0.868

405

0.405

405

7.31

S= solution of sample, DEW= Dry extract weight, A= Absorbance, GAE= Gallic acid equivalent,

TPC= total phenolic content, M= Mean

 

 

Graph 2- Graph of Standard (Quercetin) for Total flavonoidal content

Table 2. Total flavonoids content in quercetin equivalent (mg/g) of H. indicum.

S g/ml

D.E.W

(g)

A

QE conc.

C g/ml

QE conc.

C mg/ml

TFC

mg/ g

M

SEM

1000

0.001

0.569

658

0.658

658

540

1000

0.001

0.436

492

0.492

492

1000

0.001

0.418

470

0.470

470

59.34

S= solution of sample, DEW= Dry extract weight, A= Absorbance, QE= Quercetin equivalent,

TFC= total flavonoids content, M= Mean

 

Table 1 and 2 represent the total phenolic and flavonoidal content of root extract of Heliotropium indicum. Phenolic content was expressed in terms of Gal1ic acid equiva1ent (mg/g of dry weight). Flavonoidal content was expressed in terms of mil1igram of quercetin equiva1ents/g dry weight. Experiment was performed in triplet. The result was expressed in Mean SEM. The result in table 1 make known that 400 7.31 Gal1ic acid equiva1ent (mg/ g of dry weight) of phenol was present the methanolic root extract of H. indicum. The result in table 2 make known that 540 59.34 mil1igram of quercetin equiva1ents / g dry weight of flavonoid was present the methanolic root extract of H. indicum.

 

Result of table 3 indicates that % inhibition during DPPH model by methanolic root extract of H. indicum at 200 g/ml was 43. At 400g/ml the % inhibition was 58. At 600 g/ml the percentage inhibition was 75. At 800 g/ml 85 % inhibition was reported. 91 % inhibition was reported at 1000 g/ml. So, from the data we can now be able to understand that scavenging of free radical by H. indicum extract is increasing as the concentration increases. The IC50 for the methanolic root extract of H.indicum was found 268g/ml.

 

Table 3. Absorbance of different concentration of methanolic root extract of H. indicum

Sl.No.

Concentration (g/ml)

Absorbance

1

200

1.300

2

400

0.950

3

600

0.562

4

800

0.325

5

1000

0.189

6

CONTROL

2.284

 

Single or many unpaired e-s are present in free radicals. These are extremely unstable. They can achieve their stability by taking e- from other molecule. The result showed that methanolic root extract of H. indicum possesses significant antioxidant property. In various studies it was stated that many plants possesses potent free radical scavenging property because of its phenolic and flavonoidal component19. Phenolic compound have ability to transfer e- or can donate hydrogen20. Phenolic complex are acknowledged with redox quality which assist in donation of hydrogen, make them reducing agents and quenchers of singlet oxygen. Additionally, they also reveal potentiality toward chelation of metal. Polyphenols on next side have redox qualities that participate in neutralization of free radicals21. This ability makes it a potent scavenger of free radical. These qualities of active component in plants could result in its antioxidant activity.

 

CONCLUSION:

A considerable relation was established among the antioxidant actions and total flavonoid and pheno1ic contents demonstrating that these phyto-elements are the chief provider of antioxidant capability of H. indicum. The outcome of the current work proposed that methanolic root extract of H. indicum comprised chief compounds with antioxidant active, which if appropriately and broadly studied could afford new pool of biologically effective drug contenders in averting or curing medical condition originate because of oxidative stress. Therefore, the root extract of Heliotropium indicum is considered as secured natural supply of antioxidants can be utilized in dietary or pharmaceutical ground for the averting diseases mediated by free radicals.

 

REFERENCES:

1.        Raman, W, and Khalid, A. right Nin. Studies on free radicals, antioxidants, and co-factors. Clinical Interventions in Aging. (2007). , 2(2), 219-36.

2.        Jurnek, I, and Bezek, . S. Controversy of Free Radical Hypothesis: Reactive Oxygen Species- Cause or Consequence of Tissue Injury? Gen Physiol Biophys (2005), 24, 263-278 263. (2005). , 24, 2263-278.

3.        Stocker R, Keaney JF Role of oxidative modifications in atherosclerosis. Physiol. Rev. 2004; 84: 1381-1478.

4.        Grassi D, Desideri G, Tiberti S, Ferri C Oxidative stress endothelial dysfunction and prevention of cardiovascular diseases. Agro Food Industry Ii-tech. 2009; 20: 76-79.

5.        Devasagayam TPA, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS and Lele RD Free radicals and antioxidants in human health: Current status and future prospects. J. Assoc. Physicians India2004; 52:794804.

6.        Lee, D, Lim, B, Lee, Y. K, and Yang, H. C. Effects of hydrogen peroxide (H2O2) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines. Cell Biol Toxicol 22. (2006). , 22, 39-46.

7.        Manolagas, S, and Parfitt, A. What old means to bone. Trends Endocrinol Metab. 2010;Epub (2010). Mar 11., 21(6), 369-3744.

8.        Mate, J. M, and Perez-gomez, C. Nunez de Castro I. Antioxidant Enzymes and Human Diseases. Clinical Biochemistry. (1999). , 32(8), 595-603.

9.        Rao, L. Lycopene and the Prevention of Osteoporosis. AV R, editor. Scotland: Caledonia Science; (2006).

10.      Willcox, J, Ash, S, and Catignani, G. Antioxidants and prevention of chronic disease. Crit Rev Food Sci Nutr. (2004). , 44, 275-95.

11.      Rao, A, and Carotenoids, L. G R. and human health (Review). Pharmacological Research. (2007). , 55(3), 207-16.

12.      Dash KG, Abdullah SM. A Review on Heliotropium indicum L. (Boraginaceae). International Journal of Pharmaceutical Sciences and Research. 2013; 4(4):1253-1258.

13.      Das Kumar Ajit, Dutta KB, Sharma DG. Medicinal plants used by different tribes of Cachar district, Assam. Indian Journal of Traditional Knowledge. 2008; 7(3):446-454.

14.      Kumar Pandi P, Ayyanar M, Ignacimuthu S. Medicinal plants used by Malasar tribes of Coimbatore district, Tamil Nadu. Indian Journal of Traditional Knowledge.2007; 6(4):579-582.

15.      Michalak A Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Pol. J. Environ. Stud.2006; 15: 523-5.

16.      McDonald S, Prenzler PD, Autolovich M, Robards K Phenolic content and antioxidant activity of olive extracts. Food Chem. 2001; 73:73-84.

17.      Dewanto V, Wu X, Adom KK, Liu RH Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. Journal of Agricultural and Food Chemistry 2002; 50:30103014.

18.      Sreejayan N and Rao MNA Free radical scavenging activity of curcuminoids. Drug Res. 1996; 46: 169-178.

19.      Rice-Evans CA, Miller NJ, Bolwell PG, Gramley PM, Pradham JB The relative antioxidant activities of plant derived polyphenolic flavonoids. Free Radical Res. 1995; 22:375-383.

20.      Yokozawa T, Chen CP, Dong E, Tanaka T, Nonaka GI, Nishioka I Study on the inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2-picrylhydrazyl radicals. Biochem Pharmacol 1998; 56: 213-222.

21.      Obayed Ullah M, Sultana S, Haque A, Tasmin S., (2009), Eur J Sci Res, Antimicrobial, cytotoxic and antioxidant activity of Centella asiatica, 3, 2009, 260-264.

 

 

 

 

 

Received on 14.12.2019 Modified on 13.02.2020

Accepted on 01.04.2020 RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):235-238.

DOI: 10.5958/0974-360X.2021.00041.X