In Vitro Pharmacological activity of Zinc and Copper ...joics.org/gallery/ics-3567-.pdf · In Vitro Pharmacological activity of Zinc and Copper nanoparticles using medicinal plant

In Vitro Pharmacological activity of Zinc and Copper

nanoparticles using medicinal plant of Acalypha indica

root extracts.

N. Muruganantham*1 and P. Anitha2

*1 PG & Research Department of Chemistry, Thanthai Hans Roever College (Autonomous),

(Affiliated to Bharathidasan University), Perambalur - 621220, Tamil Nadu, India. 2Department of Physics, Roever College of Engineering and Technology, (Affiliated to Anna

University), Perambalur - 621220, Tamil Nadu, India.

*Corresponding author: [email protected]

Abstract

Zinc and Copper nanoparticles are synthesized from aqueous extracts of Acalypha indica root.

Pharmacological activitis such as Anti-oxidant, Anti- inflammatory activity and Anti diabetic

activity were studied. The ZnNPs and CuNPs have showed significant antibacterial activity on

multi drug resistance in biological fields. The ZnNPs and CuNPs had shown strong antioxidant

by DPPH scavenging activity. The ZnNPs and CuNPs exhibited strong anti-inflammatory

activity by albumin denaturation activity. The ZnNPs and CuNPs had strongly inhibited the

aglucosidase to a-amylase. To the best of our knowledge, this is the first attempt on the

synthesis of ZnNPs and CuNPs nanoparticles using Acalypha indica root extracts. Hence, to

validate our results, the in vivo studies at molecular level are needed to develop an antioxidant,

anti-diabetic and anti-inflammatory agent. Results of the present study, highlighted the eco-

friendly approach of plant mediated synthesis of nano particles and its potential application in the

field as an alternative to chemical drug for disease management.

Keywords: Zinc and Copper nanoparticles, antioxidant, anti-diabetic and anti-inflammatory

agent etc.,

1. Introduction

Medicinal herbs are moving from fringe to mainstream use with a greater number of

people seeking remedies and health approaches free from side effects caused by synthetic

Journal of Information and Computational Science

Volume 10 Issue 4 - 2020

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mailto:[email protected]

chemicals. India officially recognizes over 3000 plants for their medicinal value. It is generally

estimated that over 6000 plants in India are in use in traditional, folk and herbal medicine. This

article aims to provide a comprehensive review on the pharmacological aspects of Acalypha

indica. It is obtained from deciduous and mixed-monsoon forests throughout greater parts of

India.

Over the last few years, researchers have aimed at identifying and validating plant-

derived substances for the treatment of various diseases. Interestingly it is estimated that more

than 25% of the modern medicines are directly or indirectly derived from plants. It is worth

mentioning that Indian medicinal plants are considered as a vast source of several

pharmacologically principles and compounds that are commonly used as home remedies against

multiple ailments [1].

Indian traditional medicine is based on various systems including Ayurveda, Siddha,

Unani and Homoeopathy. The evaluation of these drugs is primarily based on phytochemical,

pharmacological and allied approaches including various instrumental techniques such

chromatography, microscopy and others. With the emerging worldwide interest in adopting and

studying traditional systems and exploiting their potential based on different health care systems,

the evaluation of the rich heritage of traditional medicine is essential [2].

In this regard, one such plant is Acalypha indica L. Acalypha indica has been extensively

used in Ayurvedic system of medicine for various ailments. It is deciduous and mixed-monsoon

forests throughout greater parts of India, is widely used in traditional medicinal system of India

has been reported to possess hepatoprotective, anti-inflammatory, antitussive, antifungal and

used also check wounds healing and antibacterial [2].



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Taxonomic Classification

Fig.1 Acalypha indica Fig.2 Acalypha indica

(Root)

Table.1 Taxonomic Classification

Among all the noble metal nano particles, silver nanoparticles have gained interests

because of their unique properties such as chemical stability, good conductivity, catalytic and

most important antibacterial, antiviral, antifungal and anti-inflammatory activities [3]. The wide

applications of silver nano particles has opened up another arena of research interest that is green

nanotechnology or biosynthesis of nanoparticles utilizing algae, actinomycetes, bacteria, fungi

and plants. Among the various biological synthesis procedures plant mediated synthesis of silver

nanoparticles has attracted more attention nowadays. Synthesis of silver nanoparticles has been

investigated utilizing many plant extracts like Marigold flowers [4], leaf extract of Acalypha

indica [5]

The silver nanoparticles are the most concentrated interest for research due to its potential

in applications like to transform the physical, electronic and optical properties of a compound,

treatment of cancer, as medical devices, antimicrobial coatings of paint, textiles, also biological

Kingdom : Plantae

Class : Magnoliopsida

Order : Euphorbiales

Family : Euphorbiaceae

Subfamily : Acalyphoideae

Genus : Acalypha

Species : Acalypha indica Linn.



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or chemical sensing, also possess anti inflammatory, antiviral, antifungal and antiplatelet

activities [6,7]. In this present study, a plant extract of Acalypha indica are used, which grows in

wastelands, gardens, and roadside. They are herbaceous annual weed that grows up to a height of

15 m. It falls under the family of Euphorbiaceae, which has diuretic, antimicrobial, anti-

helminthic properties and used in the treatment of respiratory problems like bronchitis,

pneumonia, and asthma [8].

This plant is used as diuretic, antihelmintic and for respiratory problems such as

bronchitis, asthma and pneumonia [9]. The roots of A. indica is used as laxative and leaves for

scabies and other cutaneous diseases [10]. Major phytochemicals identified from A. indica are

acalyphine, cyanogenic glycoside, inositol, resin, triacetomamine and volatile oils [11]. This

plant has been used extensively in herbal medicine in many tropical and sub tropical countries

[12,13]. A. indica having analgesic and anti-inflammatory effects. In Malaysia, A. indica is used

for generations for the treatment of superficial fungal and several other bacterial infections

[14].Previous studies on A. indica revealed that this plant has antibacterial activity against

several gram positive bacteria [15,16].

Gold nanoparticles of size 20-30 nm were rapidly synthesized using aqueous leaves

extract of Acalypha indica as novel source of bio-reductants [17]. Incidentally, biological

systems have long been known to reduce metal ions into nano particles [18] and many

researchers have recently reported the biogenic synthesis of silver and gold nano size

particles using a wide range of biological resources like bacteria [19]fungi [20]and plants [21]n

the plant mediated green chemistry approach, the reduction rate of metal salts is very fast and the

procedure itself requires no specific conditions unlike the physical and chemical methods [22,23]

Besides, this biogenic method of nanoparticles are synthesis appears to be reproducible and the

particles, produced through this environmentally friendly approach, are found highly stable [24-

26].

In the study, the silver nanoparticles were synthesized from these plant extracts, and

checked for their Anti-oxidant, Anti- inflammatory activity and Anti diabetic activity.

2. Materials and methods.

2.1 Collection of root

Fresh root of the samples were collected from Perambalur, during the month of December.



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https://www.sciencedirect.com/topics/engineering/nanosized-particle

https://www.sciencedirect.com/topics/engineering/gold-nanoparticles

https://www.sciencedirect.com/topics/engineering/gold-nanoparticles

https://www.sciencedirect.com/topics/engineering/nanoparticle-synthesis

2.2 Preparation of root extract

The fresh and young root of the samples were collected & washed thoroughly with sterile double

distilled water (DDW). Twenty grams of sterilized root samples were taken and cut into small

pieces. Finely cut roots were placed in a 500 ml Erlenmeyer flask containing 50ml of sterile

DDW. After that, the mixture was transferred to Soxhlet apparatus to derive extracts. The extract

was stored in 4 0C.

2.3 Microwave assisted synthesis of metal nanoparticles

Metal nitrate was used as precursor in the synthesis of metal nanoparticles. 100 ml of root extract

was added to 100 ml of 0.1N metal nitrate aqueous solution in conical flask of 250 ml content at

room temperature. The flask was thereafter put into shaker (100 rpm) at 500 C and reaction was

carried out for a period of 12 hrs. Then the mixture is kept in microwave oven for exposure of

heat.

Metals nanoparticles are made by a chemical reduction of a metal salt in the presence of a

stabilizing agent. Rapid microwave heating and agitation gives monodispersed particles. Add

200 ml of extract with 1M metal nitrate in beaker and Cover loosely. Expose the sample in

Microwave radiation for 20 minutes at 100% power. The setting of time is done on the basis of

trial and error method. The color will continue to change with respect to time. The mixture was

completely dried after a period of 20 minutes and hence nano particles in form of powders were

obtained.

2.4 Anti-Oxidant Studies

DPPH scavenging assay

The ability to scavenging the stable free radical, DPPH was measured as a decrease in

absorbance at 517 nm by the method.

Reagents

2,2-Diphenyl-1-picryl hydrazyl (DPPH) – 90.25mM in methanol in a dark room.

Procedure

To a methanolic solution of DPPH (90.25 mM), an equal volume of ethanolic Rhizome of

Cyperus rotundus L (250-1500 µg) was added and made up to 1.0 mL with methanolic DPPH.

An equal amount of methanol was added to the control. After 20 min, the absorbance was

recorded at 517 nm in a Systronics UV-visible Spectrophotometer. Ascorbic acid was used as



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standard for comparison. The inhibition of free radicals by DPPH in percentage terms (%) was

calculated by using the following equation.

% Scavenging = (OD of Control- OD of Sample/ OD of Control) X 100.

Where A control is the absorbance of the control reaction (containing all reagents except the test

compound), and A sample is the absorbance of the test compound.

2.5 Anti- inflammatory activity

Inhibition of Albumen Denaturation

The reaction mixture was consisting of test extracts and 1% solution of bovine albumin fraction,

pH of the reaction mixture was adjusted using small amount at 37°C HCl. The sample extracts

were incubated at 37°C for 20 minutes and then heated to 51°C for 20 minutes after cooling the

samples the turbidity was measured spectrophotometrically at 660 nm. Diclofenac sodium was

taken as a standard drug. The experiment was performed in triplicates. Percent inhibition of

protein denaturation was calculated as follows:

Percent inhibition (%) = (OD of Control- OD of Sample/ OD of Control) X 100.

2.6 Inhibition Of Alpha-Amylase Enzyme

Starch solution (0.1% w/v) was prepared by stirring 0.1 g of potato starch in 100 ml of 16 mM of

sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of α-amylase in

100 ml of distilled water. The colorimetric reagent is prepared by mixing sodium potassium

tartarate solution and 3,5-di nitro salicylic acid solution 96 mM. The starch solution is added to

the both control and plants extract tubes and left to react with α-amylase solution, under alkaline

conditions at 25ºC. The reaction was allowed for 3 min. The generation of maltose was

quantified by the reduction of 3,5-dinitro salicylic acid to 3-amino-5-nitro salicylic acid. This

reaction is detectable at 540 nm.

% Inhibition = (OD of Control- OD of Sample/ OD of Control) X 100.



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3. Result and Discussion

3.1 Anti – Oxidant activity

DPPH scavenging assay method

There are several methods available to assess the antioxidant activity of compounds. DPPH free

radical scavenging assay is an easy, rapid, and sensitive method for the antioxidant screening of

plant extracts. In the presence of an antioxidant, DPPH radical obtains one more electron and the

absorbance decreases.

In the present study, the ZnNPs and CuNPs using root extracts of Acalypha indica high

DPPH scavenging capacity, which increased with increasing concentration [Table 2 and Figure

3]. It is evident from the data presented in Table, that the sample possesses DPPH assay activity.

For the ZnNPs, the result shows the percentage of cytotoxicity for 250 mg/ml as 10.09%, 500

mg/ml as 32% and 750 mg/ml as 49.48%. For the CuNPs, the result shows the percentage of

cytotoxicity for 250 mg/ml as 41.25%, 500 mg/ml as 51.91% and 750 mg/ml as 72.67%. These

inhibition values are compared with standard drug of Ascorbic acid for 250 mg/ml as 23.63%,

500 mg/ml as 29.00% and 750 mg/ml as 45.25%.

The DPPH assay was carried out at different concentrations of ZnNPs and CuNPs using

root extracts of Acalypha indica, namely 250 mg/ml, 500 mg/ml and 750 mg/ml. DPPH assay

did not show any significant difference at 250 mg/ml Concentrations in Acalypha indica,

however, it was significant for 500 mg/ml and 750 mg/ml for the nanoparticles, all the values are

compared with standard drug of Ascorbic acid. Hence, this assay provided information on the

reactivity of test samples with a stable free radical.

As a part of the investigation on the mechanism of the anti‐oxidant activity, ability of

extract to inhibit DPPH scavenging assay was studied. The in-vitro study of anti‐oxidant activity

indicates that the inhibition percentage of DPPH scavenging assay by Acalypha indica root

extracts of CuNPs activity is higher than ZnNPs.



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S.No Test

Concentration

of the sample

(mg/ml)

% of

inhibition of

the ZnNPs

% of

inhibition of

the CuNPs

Ascorbic

acid

(Standard)

1

DPPH

250 10.09 41.25 23.63

2 500 32 51.91 29.00

3 750 49.48 72.67 45.25

Table.2 Anti Oxidant activity of ZnNPs and CuNPs using root extracts of Acalypha indica

by DPPH Scavenging assay.

Fig.3 Graphical representation of Anti oxidant activity of ZnNPs and CuNPs using root

extracts of Acalypha indica by DPPH Scavenging assay.

3.2 Anti- inflammatory activity

Inhibition of Albumen Denaturation method

There are certain problems in using animals in experimental pharmacological research, such as

ethical issues and the lack of rationale for their use when other suitable methods are available.

Hence, in the present study, the protein denaturation bioassay was selected for in vitro

assessment of the anti-inflammatory property of Cobalt nanoparticles synthesized Acalypha

0

10

20

30

40

50

60

70

80

250 500 750

% o

f in

hib

itio

n

Concentration

DPPH Scavenging assay activity

% of inhibitionof the ZnNPs

% of inhibitionof the CuNPs

Ascorbic acid(Standard)



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indica. The Albumen Denaturation is a well-documented cause of inflammation. Most biological

proteins lose their biological functions when denatured. Production of autoantigen in certain

arthritic disease is due to denaturation of protein. The mechanism of denaturation involves an

alteration in electrostatic hydrogen, hydrophobic, and disulfide bonding. In the present study,

denaturation of proteins is the main cause of inflammation. As part of the investigation on the

mechanism of the anti-inflammatory activity, ability of the extract to inhibit protein denaturation

was studied. Selected extracts were effective in inhibiting heat-induced albumin denaturation.

Aspirin was used as a standard anti-inflammation drug as shown in Figure [Table 3 and Figure

4]. The albumin denaturation method was carried out for ZnNPs and CuNps at different

concentrations such as 100µg/ml 200µg/ml and 300 µg/ml.

For the ZnNPs, the result shows the percentage of cytotoxicity for 100 mg/ml as 43.07%,

200 mg/ml as 52.20% and 300 mg/ml as 58.02%. For the CuNPs, the result shows the percentage

of cytotoxicity for 100 mg/ml as 48.04%, 200 mg/ml as 58.08% and 300 mg/ml as 64.32%.

These inhibition values are compared with standard drug of Aspirin

for 100 mg/ml as 45%, 200 mg/ml as 56.25% and 300 mg/ml as 66.20%. Albumen Denaturation

did not show any significant difference at 100 mg/ml Concentrations in Acalypha indica,

however, there was a significant change when the concentrations are 200 mg/ml and 300 mg/ml

for the nanoparticles and all the values are compared with standard drug of Aspirin.

As a part of the investigation on the mechanism of the anti‐oxidant activity, ability of

extract to inhibit Inhibition of Albumen Denaturation was studied. The in-vitro study of Anti-

nflammatory activity indicates that the inhibition percentage of Albumen Denaturation by

Acalypha indica root extracts of CuNPs activity is higher than ZnNPs.



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S.No Test

Concentration

of the sample

(mg/ml)

% of Protein

Denaturation

of the ZnNPs

% of Protein

Denaturation

of the CuNPs

Aspirin

(Standard)

1

Albumin

denaturation

100 43.07 48.04 45

2 200 52.20 58.08 56.25

3 300 58.02 64.32 66.20

Table.3 Anti-inflammatory activity of ZnNPs and CuNPs using root extracts of Acalypha

indica by Albumen Denaturation.

Fig.4 Graphical representation of Anti-inflammatory activity of ZnNPs and CuNPs using

root extracts of Acalypha indica by Albumen Denaturation.

3.3 Anti diabetic activity

Inhibition of Alpha-Amylase Enzyme

Diabetes mellitus is a group of metabolic diseases in which there are high blood sugar

levels over a prolonged period. A therapeutic approach to decrease the hyperglycaemia is to

0

10

20

30

40

50

60

70

100 200 300

% o

f in

hib

itio

n

Concentration

Albumen Denaturation

% of Protein Denaturation of the ZnNPs

% of Protein Denaturation of the CuNPs

Aspirin(Standard)



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inhibit the carbohydrate digesting enzymes (α-glucosidase and α-amylase), thereby preventing

the breakdown of carbohydrates into monosaccharides which is a main cause of increasing blood

glucose level. Therefore, developing compounds having inhibitory activities towards

carbohydrate hydrolysing enzymes may be a useful way to manage diabetes. As shown in Figure

4 and Table 5, α-amylase and α-glucosidase were significantly inhibited in a dose-dependent

manner by the ZnNPs and CuNPs. The results suggest that with the increased ZnNPs and CuNPs

concentration, the activity levels of enzyme were remarkably reduced, Hence, the biomolecules

likely enhanced the antidiabetic potential of the synthesized NPs. α-Amylase inhibitory actions

were observed in increasing order, as Acarbose (Figure 5). Comparable results were observed.

However, the foregoing results suggest that the synthesized ZnNPs and CuNPs have potential

antidiabetic property and could prove its effectiveness in the diabetes care.

For the ZnNPs, the result shows the percentage of cytotoxicity for 0.05 mg/ml as 57.03%,

0.1 mg/ml as 60.06% , 0.15 mg/ml as 61.02%, 0.2 mg/ml as 64.02% and 0.25 mg/ml as 60.03%.

For the CuNPs, the result shows the percentage of cytotoxicity for 0.05 mg/ml as 34.02%, 0.1

mg/ml as 43.02% , 0.15 mg/ml as 48.02%, 0.2 mg/ml as 60.02% and 0.25 mg/ml as 61.05%.

These inhibition values are compared with standard drug of Acarbose

for 0.05 mg/ml as 35%, 0.1 mg/ml as 42% , 0.15 mg/ml as 56%, 0.2 mg/ml as 61% and 0.25

mg/ml as 79%.

Albumen Denaturation did not show any significant difference at 0.5 µg/ml and

0.10µg/ml, however, it showed a significant change when the concentration of nanoparticles are

0.15µg/ml, 0.20µg/ml and 0.25 µg/ml concentrations. All the values are compared with standard

drug of Acarbose (Figure 5). On comparing, it was observed that when the concentration of the

sample increases the inhibition also increases showing a good sign of Anti-diabetic activity.

As a part of the investigation on the mechanism of the Anti diabetic activity, ability of

extract to inhibit Inhibition of Alpha-Amylase Enzyme was studied. The in-vitro study of Anti

diabetic activity indicates that the inhibition percentage of Alpha-Amylase Enzyme by Acalypha

indica root extracts of ZnNPs activity is higher than CuNPs.



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Table.4 Anti diabetic activity of ZnNPs and CuNPs using root extracts of Acalypha indica

by Alpha amylase.

Fig.5 Graphical representation of Anti diabetic activity of ZnNPs and CuNPs using root

extracts of Acalypha indica by Alpha amylase.

0

10

20

30

40

50

60

70

80

90

0.05 0.1 0.15 0.2 0.25

% o

f in

hib

itio

n

Concentration

Inhibition of Alpha-Amylase Enzyme

% of inhibitionof the ZnNPs

% of inhibitionof the CuNPs

Acarbose(Standard)

S.No Test

Concentration

of the sample

(µg/ml)

% of inhibition

of the ZnNPs

% of

inhibition of

the CuNPs

Acarbose

(Standard)

1

Alpha amylase

inhibitory

activity

0.5 57.03 34.02 35

2 0.1 60.06 43.02 42

3 0.15 61.02 48.02 56

4 0.2 64.02 60.02 61

5 0.25 60.03 61.05 79



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4. Conclusion

Scientists have shifted their interest from chemical or physical methods to biological methods as

it does not involve a combination of abusive or toxic chemicals to human health or any

involvement of immense machines or equipment. The biological methods incorporate other plant

or microbial mediated methods that are cheap and easily accessible in daily life. The medicinal

plant Acalypha indica has been used as a traditional medicinal plant due to the presence of

phytochemicals in it. The various applications of the root extract have already been established

till date. Now, in this study, the root extracts have been used for the biogenesis of the ZnNPs and

CuNPs nanoparticles. The DPPH assay is the most acceptable, fastest and simplest method for

the calculation of the free radical scavenging activity. As shown in the Table 2 and Figure 3. the

ZnNps and CuNps shows better antioxidant property when compared with the standard ascorbic

acid with an IC50 values. Denaturation of proteins is a well-documented cause of inflammation.

Phenylbutazones, salicylic acid, flufenamic acid (anti‐inflammatorydrugs), have shown dose

dependent ability to thermally induced protein denaturation. As a part of the investigation on the

mechanism of the anti‐inflammatory activity, ability of extract to inhibit protein denaturation

was studied. The in-vitro study of a anti-inflammatory activity indicates that the inhibition

percentage of albumin denaturation by Acalypha indica Root extracts. It is inferred that the

Anti- inflammatory activity of CuNPs synthesized from Acalypha indica root extracts indicates

a good and higher inhibition percentage than ZnNPs from Acalypha indica root extracts as

presented in Table 3 and Figure 4. α-amylase is a key enzyme in carbohydrate metabolism.

Inhibition of α-amylase is one of the strategies for treating diabetes. Amylase inhibitors are also

known as starch blockers because they contain substances that prevent dietary starches from

being absorbed by the body. Amylase inhibitor with starchy meal will reduce the usual rise in

blood sugar levels. The result suggests that Zn Nanoparticle exhibits well α- amylase inhibition

under in vitro conditions (Tables 4 and Fig 5).



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