Evaluation of Anti-inflammatory and Anti-arthritic activity of Luffa acutangula peel extract mediated ZnO nanoparticles

 

R. Ananthalakshmi1*, S. R. Xavier Raja Rathinam2, A. Mohamed Sadiq3

1Biochemistry Division, National Sugar Institute, Kanpur, Uttar Pradesh, India.

2Department of Chemistry, Sacred Heart College, Tirupattur, Vellore District, Tamilnadu, India.

3Department of Biochemistry, APCAS, Kalavai, Vellore District, Tamilnadu, India.

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

 

ABSTRACT:

The special improved characteristics and different application of zinc oxide nanoparticles made them to play a major role among metal oxide nanoparticles. Zinc oxide nanoparticles were successfully synthesized in a cost effective, time saving and safety method using Luffa acutangula peel extract. Zinc oxide nanoparticles showed maximum absorption peak at 377.77nm in UV visible absorption spectroscopic studies. The fourier transform infrared spectroscopy (FTIR) studies revealed the presence of various functional groups attributed by aqueous peel extract along with the zinc oxide nanoparticles. The shape of the zinc oxide nanoparticles was found to be spherical under scanning electron microscopy and they were found to be agglomerated. The zinc oxide nanoparticles prepared using Luffa acutangula showed anti-inflammatory activity when studied by Human RBC membrane stabilization method. Also they effectively inhibited the egg albumin and bovine serum albumin denaturation by which they are confirmed to have anti-arthritic activity.

 

KEYWORDS: Zinc oxide nanoparticles; Luffa acutangula; Characterization; Anti-inflammatory and Anti-arthritic.

 

 


1. INTRODUCTION:

Nanotechnology provides greater contributions in the field of medicine and other clinical conditions1,2. Metallic nanoparticles represent an important area of research as their unique properties can be tuned for the desired applications3. Green chemistry-based metal nanoparticles synthesis is an area, which has gained much importance due to their non- toxicity and monodispersed nanoparticle preparation methodology. Among green synthesis methods, plants are considered as efficient candidate for nanoparticle synthesis4. Zinc oxide nanoparticles (ZnO NPs) are assumed to be the main important nanoparticle mostly because of their surface area to volume ratio increase and are used in various engineering and science fields such as catalysis, electrochemistry, textile industry, cleaning agents, medical devices, etc.,5,6

 

ZnO NPs, produced by green route, are effective in antimicrobial, photocatalytic, and anticancer activity in comparison to ZnO NPs which are prepared conventionally7. ZnO nanoparticles are considered as a biosafe material for biological species8.

 

Multiple types of inflammations caused by foreign pathogens or chemicals and mutations that up regulate inflammation enhancers kindle the need of developing new vectors for the treatment of inflammatory diseases9. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of rheumatism diseases, such as rheumatoid arthritis and pain. In spite of their extensive usage; NSAIDs are associated with many adverse effects like myocardial infarction, gastric irritation, loose stool, nausea, vomiting, liver damage and dyspepsia. Non-steroidal anti-inflammatory drugs are widely used in the treatment of a number of inflammatory conditions, but gastrointestinal (GI) lesions have often limited their clinical utilization10. Nanoparticles have a better penetrating capacity in epithelial cells and inflammatory cells which leads to better effectiveness and better persistence in the treatment11. E Yadav et al.,12 reported the anti-inflammatory activity of biofabricated ZnO nanoparticles of Trianthema portulacastrum Linn.

 

The present investigation aimed at the study of invitro anti-inflammatory and anti-arthritic effect of biosynthesized zinc oxide nanoparticles.

 

2. MATERIALS AND METHODS:

2.1 Collection and preparation of Plant sample:

The Luffa acutangula was collected from the fields of Tirupattur, Vellore District, Tamilnadu, India. The plant parts were authenticated from Plant Anatomy Research Centre. Luffa acutangula obtained was washed thoroughly with double distilled water and the skin of cleaned Luffa acutangula was peeled off. The peeled skin was shade dried and was made into a fine powder using electric mixing grinder. The ground powder was sieved, stored in an air tight container and used whenever it was needed. Ten grams of Luffa acutangula peel powder was weighed and added to 100ml of double distilled water in a conical flask. This was kept in boiling water bath at 100°C for 60 minutes and allowed to cool at room temperature. Then the contents were filtered through what man No: 1 filter paper. The filtrate was used for further work13.

 

2.2 Biosynthesis of zinc oxide nanoparticles:

Zinc nitrate hexahydrate Zn(NO3)26H2O was used as a source of zinc  to synthesize ZnO nanoparticles using Luffa acutangula peel aqueous extract as in previously reported study. Added 10g of zinc nitrate crystals into 100ml of prepared Luffa acutangula peel extract in an Erlenmeyer flask. Mixed the contents thoroughly by shaking and then placed a magnetic pellet into the solution. The temperature was adjusted to 100° C in magnetic stirrer and kept for about half an hour. Made sure water was completely dried.  A greenish white powder obtained was nothing but zinc oxide nanoparticles coated with peel biomolecules. The powder was calcinated at 400°C for 2 hours.

 

2.3 Test for Anti- inflammatory activity:

The zinc oxide nanoparticles synthesized using aqueous extract of Luffa acutangula peel was studied for their anti-inflammatory activity using human RBC membrane stabilization method14. 3ml of my own blood sample was collected.The collected blood was transferred to the heparanized centrifuge tubes to prepare red blood cell suspension. The blood was washed twice with equal volume of saline. Prepared 10 % V/V blood suspension with saline (3ml of blood + 27ml of saline). Various concentration of ZnO nanoparticles in the range of 20 µg, 40µg, 60µg, 80µg and 100µg/ml of DMSO were prepared. 1ml of sample of various concentrations was added to 1ml of RBC suspension in the centrifuge tubes. 1ml of saline with 1ml of RBC suspension acted as control. The peel extract and the positive control – Diclofenac was also prepared with the same concentration range of ZnO nanoparticles. The experiment was carried out in triplicates under same condition. All the tubes were incubated for 30 minutes in the water bath at 56şC. After incubation, the tubes were cooled under running tap water. The tubes were centrifuged for 5 minutes at 2500rpm.  Then the supernatant was collected and measured the absorbance at 560nm using Shimadzu UV 1800 spectrophotometer. The percentage of membrane stabilization was equivalent to percentage of inhibition of hemolysis. Thus the % of inhibition of hemolysis by ZnO nanoparticles, peel extract and Diclofenac was calculated by

 

% Of inhibition = (Ac – As)Ac × 100

 

Where Ac is the absorbance of the control and As is the absorbance of the sample.

 

2.4 Test for Anti- arthritic activity:

Zinc oxide nanoparticles were studied for their anti arthritic activity by protein denaturation method15.

 

2.4.1 Egg albumin denaturation method:

To 0.5ml of 0.5% aqueous bovine serum albumin (BSA) added 1ml of ZnO nanoparticles of various concentrations ranging from 20 - 100µg/ml of DMSO. 0.5ml of BSA solution and 1 ml of DMSO acted as control. The peel extract and the positive control – Diclofenac was also prepared with the same concentration range of ZnO nanoparticles. The experiment was carried out in triplicates under same condition. The tubes were incubated at 37şC for 30 minutes. After bringing down the test tubes to the room temperature, 2.5ml of phosphate buffer saline of pH 6.3 was added to all the test tubes. The reaction leads to protein denaturation resulting in turbidity. The turbidity was measured spectrophotometrically at 660nm. The percentage of inhibition of protein denaturation by ZnO nanoparticles, peel extract and Diclofenac was calculated by

 

% Of inhibition of protein denaturation =

(Ac – As)Ac × 100

Where Ac is the absorbance of the control and As is the absorbance of the sample

 

2.4.2 Egg albumin denaturation method:

Varying concentration of ZnO nanoparticles in the range of 20-10µg/ml dissolved in DMSO was prepared. To 1 ml of varying concentration of nanoparticles, 2.8ml of phosphate buffered saline of pH 6.4 and 0.2ml of egg albumin solution was added. 1 ml of DMSO instead of sample served as control. The peel extract and the positive control – Diclofenac was also prepared with the same concentration range of ZnO nanoparticles. The experiment was carried out in triplicates under same condition. The tubes were incubated at 37şC for 15 minutes in BOD incubator. After 15 minutes of incubation, the tubes were heated to 70şC for 5 minutes. The tubes were cooled down and the absorbance was measured at 660 nm by using Shimadzu UV 1800 spectrophotometer. The percentage of inhibition of protein denaturation by ZnO nanoparticles, peel extract and Diclofenac was calculated by

 

% Of inhibition = (Ac – As)Ac × 100

 

where Ac is the absorbance of the control and As is the absorbance of the sample.

All the methods were carried out in accordance with relevant guidelines and regulations.

 

3. RESULTS AND DISCUSSION:

3.1 Biosynthesis of ZnO nanoparticles:

Zinc oxide nanoparticles were synthesized from zinc nitrate hexahydrate using aqueous extract of Luffa acutangula peel. The phytocomponents present in the Luffa acutangula peel extract helps in the formation of zinc oxide nanoparticles from zinc nitrate hexahydrate precursor. The   calcination process helps in improving crystallinity of zinc oxide nanoparticles synthesized16.

 

3.2 Anti- inflammatory activity:

3.2.1 Human Red Blood Cell Stabilization method:

Anti-inflammatory activity of biosynthesized ZnO nanoparticles was studied by Human RBC membrane stabilization method. The release of hydrolytic enzymes from lysosomes due to lysosomal membrane damage, leads to inflammatory disorders. Human RBC membrane was considered to be similar to lysosomal membrane17, 18. When a drug or substance is capable of preventing RBC membrane from damage either induced by heat or hyposalinity, the substance is said to possess anti-inflammatory property.

 

The inhibition of heat induced hemolysis by different concentration of ZnO nanoparticles, standard anti-inflammatory drug Diclofenac and the peel extract ranging from 20-100µg/ml was measured spectrophotometrically at 560 nm and were given in the  Table 1.

 

Table.1 Invitro anti-inflammatory activity of ZnO Nanoparticles (ZnO NPs), Diclofenac and peel extract by Human RBC membrane stabilization method.

S.

NO

Concentration in µg/ml

% of inhibition

ZnO NPs

Diclofenac

Peel extract

1

20

43.06±0.1

48.42±0.05

5±0.07

2

40

47.71±0.07

56.12±0.21

8.68±0.15

3

60

55.11±0.08

59.98±0.15

9.72±0.17

4

80

68.37±0.17

67.34±0.16

11.24±0.07

5

100

73.9±0.10

72.47±0.27

13.79±0.11

 

When human blood containing RBC is heated, they undergo hemolysis. The percentage of inhibition of hemolysis of human RBC on adding ZnO nanoparticles, was confirmed with the decrease in the optical density measured spectrophotometrically. The percentage of hemolysis inhibition exerted by minimum concentration 20µg of ZnO nanoparticles was 43.06%. The percentage of inhibition of hemolysis increases gradually with the increase in the concentration of ZnO nanoparticles. 50% hemolysis inhibitIon (IC50) was achieved by 41.57µg concentration of ZnO nanoparticles. The standard drug diclofenac showed IC50 concentration at 23.41µg.  Initially that is at 20µg Diclofenac showed good inhibition, but at 100µg ZnO NPs showed better inhibitory than standard drug. On the other hand the aqueous peel extract as such have very less hemolysis inhibition activity.

 

The inhibition of hemolysis is related to the RBC membrane stabilization. Thus, the ZnO nanoparticles are found to be capable of stabilizing RBC membrane and thereby they can effectively act as an anti-inflammatory agent.

 

Bepari et al.,19 studied the anti-inflammatory activity of the silver nanoparticles prepared using Anacardium occidentale leaf extract and reported 67.49% of hemolysis inbition with 100µg of silver nanoparticles. The ZnO nanoparticles prepared using Luffa acutangula peel extract shows 73.9% inhibition with 100µg concentration of ZnO nanoparticles.  So, ZnO nanoparticles can be recommended for management of inflammatory disorders.

 

3.3 Anti – arthritic activity:

3.3.1 Egg albumin denaturation method:

The denaturation of proteins occurs in the arthritic reaction due to tissue damage in inflammation20.

 

The inhibition of egg albumin denaturation by different concentration of ZnO nanoparticles standard drug Diclofenac and the peel extract ranging from 20-100µg/ml were measured spectrophotometrically at 660 nm and were given in the Table.2.

 

Table.2 Invitro anti-arthritic activity of ZnO Nanoparticles (ZnO NPs), Diclofenac and peel extract by Egg albumin denaturation method

S.

No

Concentration in µg/ml

% of inhibition

ZnO NPs

Diclofenac

Peel extract

1

20

45.98±0.02

47.79±0.03

2.28±0.46

2

40

55.7±0.04

57.7±0.57

5.1±0.46

3

60

62.51±0.11

63.9±0.04

7.65±0.69

4

80

75.02±0.16

75.84±0.10

11.15±0.45

5

100

78.9±0.06

84.8±0.05

12.08±0.39

 

The minimum concentration 20µg of ZnO nanoparticles, inhibited 45.98% of egg albumin protein denaturation. There was a gradual increase in the % of denaturation inhibition with the increase in the concentration of ZnO nanoparticles. The 50% inhibition of egg albumin protein denaturation was brought about by 28.07µg concentration of zinc oxide nanoparticles synthesized using aqueous Luffa acutangula peel extract. Diclofenac showed IC50 at 25.33µg/ml concentration and where as aqueous peel extract showed very minimum egg albumin denaturation inhibition.

 

Very less number of studies were carried out to study the egg albumin denaturation inhibition by nanoparticles especially ZnO nanoparticles. Kalpana nagarajan et al.,21 studied the antiarthritic activity of ZnO nanoparticles by Egg Albumin denaturation method. The egg albumin denaturation studies of ZnO nanoparticles reveal that they exhibit anti arthritic property in a significant level.

 

3.3.2. Bovine serum albumin denaturation method:

The inhibition of BSA protein denaturation at different concentration of ZnO nanoparticles, the standard drug Diclofenac and the aqueous peel extract ranging from 20-100µg/ml was measured spectrophotometrically at 660 nm and were given in the Table 3.

 

Table.3 Invitro anti-arthritic activity of ZnO Nanoparticles (ZnO NPs), Diclofenac and peel extract by Bovine serum albumin denaturation method

S.

NO

Concentration in µg/ml

% of inhibition

ZnO NPs

Diclofenac

Peel extract

1

20

50.09±0.45

52.14±0.30

3.07±0.25

2

40

63.13±0.49

61.92±0.47

6.21±0.31

3

60

66.95±0.67

72.26±0.93

9.68±0.55

4

80

71.89±0.62

79.25±1.06

12.29±0.21

5

100

81.2±0.81

85.48±0.70

16.85±0.17

 

The minimum concentration 20µg of ZnO nanoparticles, inhibited 50.09% of BSA protein denaturation. There was a gradual increase in the % of denaturation inhibition with the increase in the concentration of ZnO nanoparticles. The 50% inhibition of BSA protein denaturation was brought about by 13.14µg concentration of Zinc oxide nanoparticles synthesized using aqueous Luffa acutangula peel extract. Diclofenac showed IC50 at 11.88µg/ml concentration and whereas aqueous peel extract showed very minimum BSA protein denaturation inhibition.

 

The study conducted on the albumin denaturation inhibitory effect of ZnO nanoparticles synthesized using Pterocarpus marsupiumby Rajan et al.,22 reported the IC 50 at 98.45µg/ml. Thus, the ZnO nanoparticles synthesized using Luffa acutangula was found to be highly effective in inhibiting protein denaturation at a low concentration. Thereby it can be recommended as an antiarthritic agent for arthritis caused due to protein denaturation.

 

4. CONCLUSION:

The nanoparticles which can be synthesized using plants are fairly good due to the non - toxicity property of plants and the easy availability of plant material and are appropriate for satisfying the nanoparticle’s high demand with environmental and biomedical applications. Luffa acutangula (Cucurbitaceae), a perennial plant distributed mainly in India, is attempted to synthesis ZnO nanoparticles using its peel extract and succeeded. The synthesized ZnO nanoparticles are characterised by basic techniques and reported to be biologically active to use in pharmaceutical especially as anti-inflammatory and anti – arthritic agents.

 

5. CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

 

6. FUNDING:

This work was partially supported by Sacred Heart College, Vellore District, Tamilnadu, India, through Don Bosco Research Grant. We would like to show our gratitude to the Management of Sacred Heart College, Vellore District, Tamilnadu, India for supporting the research by granting Don Bosco Research Grant.

 

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Received on 14.04.2020            Modified on 19.05.2020

Accepted on 30.06.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):2004-2008.

DOI: 10.52711/0974-360X.2021.00355