Amir Hassan, HimayatUllah, Muhammad Israr

Page | 732

The antioxidant activity and phytochemical analysis of medicinal plant Veronica biloba

Amir Hassan1, *

, Himayat Ullah1

, Muhammad Israr2

1Department of Organic Chemistry; Government Post Graduate College Mardan (23200), Affiliated with Abdul Wali Khan University Mardan Khyber Pakhtunkhwa

Pakistan. 2Department of Botany; Government Post Graduate College Mardan (23200), Affiliated with Abdul Wali Khan University Mardan Khyber Pakhtunkhwa Pakistan.

*corresponding author e-mail address:amirhassan741@gmail.com | Scopus Author ID: 57211903251

ABSTRACT

A medicinal plant veronica genus has 450 well known species and found across both temperate and hemisphere region their 26 species are endemic and

known in a total of 79 popular species and are widely utilized throughout the world due to important biological activities. In this study fully powdered

uniform size specie veronica biloba plant taken in porous bag were manually subjected to soxhlet hot continuous process for cyclization of extraction

using ethanol (300 mL) a concentrated dried extract obtained after solvent evaporation. Furthermore, liquid-liquid extracted fractions as water,

dichloromethane, n-hexane, and ethyl acetate yields results found polar fraction with the highest percentage (water 47.51 %). The phytochemical

screening of veronica biloba has shown all major compounds entirely present in extracts. One of the primitive phenolic compound flavonoid is present

in plant and show potency towards antioxidants. All the extracted fractions of plant showed excellent antioxidant activity using a stable DPPH (1,1-

diphenyl-2-picryl-hydrazyl) method at concentration (range from 31.25 to 500µg/mL). Primary our ethyl acetate extract fraction showed the highest

inhibition potential at IC50 = 1.70±0.05µg/mL which is much closer to a standard positive control Propyl gallate showed IC50 = 1.6±0.05µg/mL percent

potential. The purification and isolation of these extract areimportant which can provide us help in novel antioxidants discovery also natural antioxidants

currently in cosmetics products, food and therapeutics health related products significantly demanded because they are very effective, efficient and

harmless as compared to synthetic.

Keywords: Soxhlet; Phytochemicals; DPPH; Antioxidants; Percent Yields.

1. INTRODUCTION

A medicinal plant veronica genus has 450 well known

species and found across both temperate and hemisphere region

[1]. In veronica plant their 26 species are endemic and known in a

total of 79 popular species [2]. Veronica species are widely

utilized throughout the world due to higher chemotaxonomic,

phytochemicals value, and important biological activities

investigated on it therefore mostly utilized by Chinese and Turkish

also in conventional or, traditional medicinal system for tonics,

wound healing, influenza, expectorants, restoratives, and also for

respiratory infection [3]. Some species of this plant areutilized in

anticancer treatment [4]. Extractions from medicinal plants

possess certain potentially active substances or, compounds which

is accountable for biological activity [5]. The most recent study

investigated in a medicinal plant founds mainly antioxidants, such

as flavonoids, Vitamin A, C, E, Lignin’s, and tannins phenolic

compounds are antioxidants and exist in plants [6]. The plants

extracted natural products give help in the new development of

pure or, standardized compounds in chemical diversity and

unknown availability [7]. The natural product is very effective,

efficient and has no or, little side effects as compared to synthetic

drugs which have side effects and is therefore people utilized

conventional drug obtained from plants [8]. The medicinal plants

possess several novels biological like antioxidants, antiviral,

antitumor, antimalarial, and antimicrobial activities and necessary

to examine these plants and are compulsory to extract the potent

compound may act as natural antioxidants or, in other activities

instead of synthetic one [9]. Currently in cosmetics products, food

and therapeutics health related products significantly demanded

the natural antioxidants having multifacetedness high amount and

mass [10]. Muchnoticeable biological activity reported on the

veronica plant species like antibacterial [11]. Mocan et al [12]

found in veronica orchidea, officinallis, teucrium, antioxidants,

phytochemical and antimicrobial. Dunkic et al [13] in veronica

spicataconfirmed antioxidants plus antimicrobial while Exarchou

[14] isolated compounds hispidulin also antiviral [15] an effective

anti-cancer, anti-mutagenic and antitumor with new compounds

[16-19] anti-ulcerogenic [20, 21] anti-cholinesterase [22] anti-

inflammatory [18] anti-insecticidal [23] anti-hepatocarcinoma [24]

antioxidants [25] angiogenic, neuroprotective potency [26] wound

healing, cough, catarrh, and kidney medication [12] natural

preservatives, dietary supplement, and hyperglycemia [27, 28] was

reported in this genus species, Javadsharif rad et al [29] found in

veronica persicapoiracetylcholinesterase, scolicidal, tyrosinase,

xanthine oxidase, lipoxygenase. Our current study is to focused

only on antioxidant analysis by (1,1-diphenyl-2-picryl-hydrazyl)

method and phytochemical confirmation also yield calculation for

the first time in the genus veronica medicinal plant a specie

veronica biloba.

2. MATERIALS AND METHODS

2.1. Material and Methods.

2.1.1. Collection and identification of the plant.

The medicinal plant veronica biloba confirmed and indentified via

a literature-survey also from Prof. Muhammad Israr botanical

export of Govt: Post Graduate College Mardan. The healthy and

fresh medicinal plant utilized in the project-experiment, plant at

flowering stage was collected during February-March months.

Selection is done from specific fertile land location Par HotiSang-

e-mar mar and RustamSurkhDheri tehsil and district 23200

Mardan. A Collected plant with roots washed with sterilized

Volume 8, Issue 4, 2019, 732 - 738 ISSN 2284-6808

Open Access Journal Received: 19.11.2019 / Revised: 20.12.2019 / Accepted: 23.12.2019 / Published on-line: 27.12.2019

Original Research Article

Letters in Applied NanoBioScience https://nanobioletters.com/

https://doi.org/10.33263/LIANBS84.732738

The antioxidant activity and phytochemical analysis of medicinal plant Veronica biloba

Page | 733

water and stabilized through air-exposure in cover and all dust

contamination along with water is removed.

2.1.2. Grinding and drying of the plant.

All the collected plant kept in a shade and avoided from light and

environmental-contamination exposure for three weeks (3-weeks).

After drying process converted into smaller pieces then equal size

powder achieved with extensivesurface area after grinding process

for a better process of extraction.

2.1.3. Soxhlet Extraction Process.

Finally uniform size fully powdered grinded 30 gram plant taken

in porous bag manually prepared from cellulose hard filter-paper

then placed in Soxhlet thimble section. In bottom flask of Soxhlet

300 mL absolute ethanol taken for cyclization of extraction

process. To condenser section water inlet-outlet wasprovided. The

mantox heater temperature maintained at 40 – 450C. The cycling

of solvent occurs through siphon arm up to 36 hrs until the

evaporated solvent does not bring any extracted residue. The dried

extract obtained after ethanol evaporation over the electrical

controllable water bath. Finally, fractions extract achieved from

after liquid-liquid extraction such as water, dichloromethane, n-

hexane, and ethyl acetate get concentrated for phytochemical

screening and for various biological analyses.

2.1.4. Phytochemicals Confirmation analysis.

The medicinal plants constitute several important biologically

active classes of compounds known as phytochemicals. Certain,

plants possess this type of characteristics the identification and

confirmation arevery useful and several studies assigned the

standard protocol for it we have used the procedure of previously

reported in [30-33] discussed given below:

2.1.5. Carbohydrates Confirmation.

Molish Test: The aqueous 3 mL freshly prepared extract taken in

test tube followed by 0.5 mL addition of Molish reagent and

slightly 2-3 drops concentrated H2SO4 added to test tube walls a

violet color ring appeared confirmed the carbohydrates presence.

2.1.6. Tannins Confirmation.

The aqueous 3 mL freshly prepared extract taken in test tube

followed by 0.5 mL addition of Ferric Chloride (FeCl3) solution a

condensed green color confirmed the tannins presence.

2.1.7. Saponins Confirmation.

Froth Test: The aqueous 3 mL freshly prepared extract taken in

test tube shaken vigorously by hand about 3 cm froth is formed in

test tube for about 5-15 minute confirmed the saponins presence.

2.1.8. Flavonoids Confirmation.

The aqueous 3 mL freshly prepared hydrochloric acid extract

taken in two test tubes one followed by 1.5 mL distilled water and

other by 1.5 mL sodium hydroxide (NaOH) addition compare both

with one another a yellow color achieved confirmed the flavonoids

presence. Another method 3 mL extract treated with 1.5 mL of

lead acetate solution a yellowish precipitate obtained positive

result indicates (flavonoids).

2.1.9. Sterols and Triterpenes Confirmation.

Lieberman’s Test: The 3 mL (EtOH) freshly prepared extract

taken in test tube followed by 0.5 mL addition of acetic anhydride

slightly 2-3 drop concentrated H2SO4 added to test tube wall a

brown-reddish color ring appear in upper layer confirmed the

sterol and triterpenes test. Salkowski Test: The 3 mL (EtOH)

freshly prepared extract taken in test tube followed by 2-3 drop

addition of concentrated H2SO4 slightly to the wall of test tube a

reddish brown color ring without greenish layer confirmed the

sterol and triterpenes presence.

2.1.10. Anthraquinone Glycosides Confirmation.

The 3 mL (EtOH) freshly prepared extract taken in test tube

followed by 1 mL addition of ammonia solution a green color in

combination with light rose color confirmed the anthraquinone

glycosides presence.

2.1.11. Resin Confirmation.

The aqueous 4.5 mL extract wasmixed with 2 mL acetone-water

of (1:1) no formation of turbidity seen in solution confirmed the

resin absence.

2.1.12. Protein and amino acid Confirmation.

Xanthoproteic Test: 3 mL extract in test tube followed by 2-3 drop

addition of concentrated nitric acid (HNO3) a yellow color

achieved confirmed the protein presence. Biuret Test: 4.5 mL

extract in test tube addition 1 mL NaOH (10 %) solution then

heated, and consequently added 0.5 mL copper-sulphate (CuSO4)

10 % solution confirmed the protein presence.

2.1.13. Tannins Confirmation.

4.5 mL extract wastreated with 1.5 mL Gelatin (1%) solution in

NaCl combination a white color precipitate achieved confirmed

the tannins presence.

2.1.14. Fats and Fixed Oils Confirmation.

Aqueous extract approximately 2.5 mL pressed between the filter

papers no seen of any oily stain confirmed the absence of fats and

fixed oils.

2.1.15. Diterpenes Confirmation.

3 mL aqueous extract in test tube wasfollowed by 2-3 drop

copper-acetate solution a bright green color obtained confirmed

presence of diterpenes.

2.1.16. Alkaloids Confirmation.

3.5 mL freshly prepared to dilute hydrochloric acidic extract

wasfollowed by 0.5 mL reagent addition of (Mayer’s, Hager and

Wagner separately added) a formation of color precipitate

indicated alkaloids presence.

2.2. Antioxidant Activity by DPPH Method.

All the dried concentrated extracted fractions (water, ethyl-

acetate, dichloromethane, and n-hexane) obtained were evaluated

for antioxidant activity using stable,DPPH (1,1-diphenyl-2-picryl-

hydrazyl) radicals scavenging power effect according to previous

[34-36]. In first all the fractions extracts dissolve in suitable

solvent taking 10 mg/mL in Eppendrops tubes and diluted by ½ in

a serial to (31.25, 62.5, 125, 250, and 500) µg/mL. The standard

DPPH (1, 1-diphenyl-2-picryl-hydrazyl) solution was made 2 hrs

prior tomethanol (100 mL) from 0.0238 gram by weight and store

in dark at room temperature covered with aluminum foil to avoid

from decomposition. In second stage the serial dilution (ranging

from 500 – 31.25 µg/mL) of each extracted fractions were mixed

separately using 10 µL of a single serial with 90 µL DPPH

solution and incubated for 30 minute at room temperature (370C),

at 517 nm absorbance was measured also in triplicates. In the

same concentration (from 31.25 to 500 µg/mL) positive controls

PG (Propyl Gallate), TBH (3-Tert-butyl-4-hydroxyanisol), and

AA (ascorbic acid) were prepared, and placed DMSO

(dimethylsulfoxide) as a negative control media. In last the percent

inhibition of free radical DPPH scavenging activity is measured

according to [37] equation.

Amir Hassan, Himayat Ullah, Muhammad Israr

Page | 734

Antioxidant activity percent % inhibition

=

Whereas Control Abs = absorbance of standard solution, and

Sample Abs = absorbance of the sample used the assay

experiments were repeated in triplicates and median inhibitory

concentration calculated using regression statistics and standard

deviation taking concentration against percent scavenging

inhibition.

3. RESULTS

3.1. Fraction Extracts Yields Calculation.

The percent yield by calculated according to [31] net dried extract

by weight obtained from after Soxhlet hot continuous extraction

process was 7.8314 (gram) about 26.10% by subjecting 30 (gram)

powdered plant to processing. Furthermore, liquid-liquid extracted

fractions yields results almost varied from one another in each

solvent. Fractions percent taken from concentrated extract

following sequence in increasing order as shown water >n-hexane

> ethyl-acetate > dichloromethane variation in percent yields are

water = 47.8%, n-hexane = 28.74%, ethyl-acetate = 20.45% and

dichloromethane = 3.28% shown in [Table 1] also in reported

literature [10, 38-42] results which is based on compounds in

plants by polarities [31] is comparable to our data and we have

found polar fraction with excellent percent yields as shown in

[Figure 1].

Figure 1. Percent-yields comparison of each concentrated fraction.

Table 1. Calculated yields values of veronica biloba for fractions extracts.

S.NO Solvent Fraction Extracts Yield Values

1 Ethyl-acetate Fraction 20.45 %

2 Dichloromethane Fraction 3.28 %

3 n-hexane Fraction 28.74 %

4 Water Fraction 47.51 %

3.2. Phytochemical Screening Confirmation.

Phytochemical screening of medicinal plant veronica

biloba evaluated according to [30-33] almost all major compounds

entirely present in extracts such as flavonoids, tannins, terpenoids,

phenol, alkaloids, protein and amino acids, glycosides and

anthraquinine, carbohydrates, saponins and sterol except fats and

oil and resin test found negative results as shown [Table 2] One of

the primitive phenolic compounds is flavonoids their presence in

plant show potency towards antioxidants [43] while steroids

shows inflammatory potential [44] and alkaloids mostly towards

antispasmodic, analgesic and antimicrobial [43-45].

Table 2. Phytochemical screening results of Veronica Biloba.

S. No Tested Phytochemical Positive

Appearance

Negative

Appearance

1 Alkaloids Positive ----

2 Flavonoids Positive ----

3 Saponins Positive ----

4 Protein and amino acids Positive ----

5 Fats and oil ---- Negative

6 Tannins Positive ----

7 Sterol Positive ----

S. No Tested Phytochemical Positive

Appearance

Negative

Appearance

8 Terpenoids Positive ----

9 Glycosides and anthraquinine Positive ----

10 Resin test ---- Negative

11 Carbohydrates Positive ----

12 Phenol Positive ----

The Veronica genus includes tannins, phenol, saponins,

carboxylic acid, steroids, and flavonoids contents due to which it

shows influential action [11]. Phytochemical are essential dynamic

compounds existing in plants example is flavonoids, alkaloids,

tannins, steroids polyphenol, terpenoids, and saponins,

significantly utilized to prescribe diseases additionally employed

in nutrient plus dietary-supplement [46-50]. Every effective

phytochemical are accountable for a biological action such as

against pathogens provide help in unlike compound development

[51-53]. Flavonoids is essentially polyphenol their bearing can

enhance the potential of the antibiotic toward microbes [54, 55].

Particular flavonoids structure complexes with the cell wall of

bacteria-protein and extracellular ingredients and is extremely

valuable plus efficient composite [56]. Some medicinally

bioactive compound appearance inside plant largely shows

antimicrobial activity possibly it bears flavonoids, tannins,

Saponins, steroids, and terpenoids [57]. Terpenoids concerned

meanwhile reducing microorganism cell wall plus membranous

tissue extinction [58]. Steroid in antimicrobial capable of liposome

leakage of lipid bilayer membrane [59].Saponins synergy amidst

microbes produces enzyme protein leakage of the cell [60]. The

confirmation of phytochemical screening in specie veronica biloba

is for first time reported.

3.3. Antioxidant Activity by DPPH.

The antioxidant activity of medicinal plant a specie

veronica biloba is evaluated according to prescribed procedure of

[34-36] to confirm whether the extracted fractions possess

hydrogen plus free radical scavenging potency because in order to

eliminate the oxidative stress and its effects to keep the organisms

[61]. All the extracted fractions of plants showed excellent

antioxidants results in comparison to the standard as shown in

[Table 3] the concentration used for activity (range from 31.25 to

500µg/mL). Primary, the ethyl-acetate extracted fraction showed

highest IC50 = 1.70±0.05µg/mL inhibition potential and is almost

very closed to the standard positive control Propyl gallate showed

IC50 = 1.6±0.05µg/mL percent potency and is near to other

standard as shown in [Figure 2]. Secondary, dichloromethane

extract fraction showed IC50 = 2.13±0.05µg/mL percent inhibition

which is also near to standard used. While other extract fractions

water showed IC50 = 6.8±0.15µg/mL and similarly n-hexane

showed IC50 = 5.57±0.1µg/mL percent inhibition and standard

ascorbic acid IC50 = 1.27±0.01µg/mL also of 3-Tert-butyl-4-

hydroxyanisol IC50 = 1.2±0.1µg/mL inhibition as shown as shown

in [Table 3]. Our current results aresimilar to [31, 62] when

The antioxidant activity and phytochemical analysis of medicinal plant Veronica biloba

Page | 735

compared we have concluded that this species possess an

antioxidant potential and is the first report on it. An unusual

balance among antioxidant maintenance and the generation of

reactive oxygen species. The oxidative extension may additionally

result in both the raised generation of reactive oxygen species or

from lack of antioxidants [alpha tocopherol, ascorbate or

glutathione]. Inside our body Free radicals mean generated if our

body cells are flashed to numerous substances like chemicals,

radiation, fumes, sun and pesticide whiskey, smoke contamination,

and also due to several metabolic processes including a deposited

fat loss to obtain energy. Irregular intake continues likewise

accountable toward the creation of free radicals. Free radicals

react with protein plus lipids and likewise with

glycosaminoglycans an extracellular matrix [For example Iduronic

acid] and produce associated diseases. Polyunsaturated fatty acids

also amino acids include sulfur are predominantly sensitive. While

the succeeding is observed in large concentrations inside the

CNS, moreover, a study on the use of free radicals in cerebral

ischemia has been conveyed on these molecules. Free radicals

frequently charge at the alpha-methylene carbons that are close to

the carbon-carbon double bond inside fatty acids. Especially

polyunsaturated fatty acids having certain dual bonds in molecules

are consequently capable of free radical impairment [63].

Figure 2. Comparison of Inhibitory Concentration (IC50 Values) Each

Extracted fraction.

Table 3. Antioxidant activity results of Veronica Biloba fraction extracts.

S. NO Fraction Extracts IC50± SD (µg/mL)

1 n-hexane Fraction 5.57 ± 0.1

2 Water Fraction 6.8 ± 0.15

3 Dichloromethane Fraction 2.13 ± 0.05

4 Ethyl-acetate Fraction 1.70 ± 0.05

5 Propyl Gallate(Positive control) 1.6 ± 0.05

6 3-Tert-butyl-4-hydroxyanisol (Positive control) 1.2 ± 0.1

7 Ascorbic acid (Positive control) 1.27 ± 0.01

The data calculated using test statistics as Mean ± SD (a standard deviation). Median inhibitory concentration

(IC50) taken percent inhibition versus different concentration via standard software programmer.

4. CONCLUSIONS

Our current study has shown that a medicinal plant

veronica biloba fraction extracts contain all the important types of

phytochemicals and exhibit the antioxidants inhibition potential.

All the extracts obtained with a good yield mainly in polar parts

and primitive phenolic compound flavonoid is present in this

plant, shows potency towards antioxidants. Our ethyl acetate

extract fraction showed highest inhibition potential at IC50 =

1.70±0.05µg/mL which is much closer to a standard positive

control Propyl gallate showed IC50 = 1.6±0.05µg/mL percent

potential. The purification, isolation and characterization of these

extract areimportant because natural antioxidants currently in

cosmetics products, food and therapeutics health related products

significantly demanded because they are very effective, efficient

and harmless.

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Amir Hassan, Himayat Ullah, Muhammad Israr

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6. ACKNOWLEDGEMENTS

Amir Hassan thanks to his supervisor Prof. Himayat Ullah of Organic Chemistry Department, Govt. Post Graduate College

Mardan, for his great support and work. Our research work not receives any specific funding from anywhere or any source in the

designing of Study interpretation and data collection analysis, writing and Publication of Manuscript.

© 2019 by the authors. This article is an open access article distributed under the terms and conditions of the

Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).