Introduction

Nanoparticles represent a particle with a nanometer size of 1–100 nm. The most widely studied nanoparticle materials are metal nanoparticles because they are easier to synthesize 1. In the present work, Lantana camara (also known as red sage) leaf extract was used to synthesize silver nanoparticles for antimicrobial studies. Some of the most studied metallic nanoparticles include silver, gold, platinum, and palladium. Ag nanoparticle is an interesting metal to be studied, especially in the field of health and medicine. Silver is a strong antibacterial and also toxic to cells 2. Silver has the ability to damage bacterial cell walls, inhibits bacterial cell growth, and disrupts cell metabolism because of the interaction between Ag ions with macromolecules in cells, such as proteins and deoxyribonucleic acid. The Ag nanoparticles are chemically more reactive than silver in their bulk. The plant Lantana camara Linn. (Verbenaceae) is an ornamental herb. It is found in South India, America, and Africa, mostly native to subtropical and tropical countries. The number of species is available with genus Lantana. Some species of Lantana are Lantana trifolia, Lantana salvifolia Jacq. and Lantana indica Roxb. These leaves are rich in various chemical constituents such as triterpenes, glycosides, steroids, flavonoids, and essential oils 3. The plant contains various medicinal properties and used in the treatment of skin diseases such as dermatitis, itching, scabies, leprosy, and chickenpox.

MATERIALS

Fresh plant of Lantana camara was collected from S.V. University, Thirupathi. Silver Nitrate purchased from Wise scientific, India. Gentamicin purchased from Aristo Pharmaceuticals, Mumbai, India. Cepftrione purchased from Ranbaxy Pharmaceuticals, Mohali, India. Diclofenac purchased from Cipla pharmaceuticals. India. All analytical grade and purchased from Hi Media (Mumbai).

Methods

Cold Extraction

Lantana Camara was separately shade dried, crushed to crude powder was macerated with ethanol and aqueous based on their polarity following cold extraction method, and filtrates were concentrated by rotary evaporator to yield was about 4% dense residues 4. Based on the phytochemical analysis and 99% ethanol and aqueous extract were selected for further experiments. Ethanol and aqueous extract of Lantana camara were prepared their anti microbial activity was done using microbial maximum inhibitory concentration.

Phytochemical Screening

Test for flavonoids: Alkaline reagent^’s, Shinoda test

Test for alkaloids: Dragendroffs test, Mayers test, Wagner's test.

Test for carbohydrates: Benedict's test, Molischs test, Fehling's test

Test for proteins: Biuret test, Xanthoproteic test, Millon's test

Test for glycosides: Liebermann’s test, Salkowskis test, Killer Killian test

Test for phenols: Ferric chloride test, Test for tannins, Gelatin solution

Test for free amino acids: Ninhydrin test, Test for quinines, HCL test; Test for the steroids: Liebermann Burchard test; Test for saponins: Foam test, Froth test 5.

In-vitro antibacterial activity

Disc diffusion method

Prepare the inoculums

10⁸ colonies forming unit per ml bacterial inoculums in a nutrient broth which is prepared by picking 3-5 isolated colonies from the plate 6. The winning streak the swab on the surface going from nutrient agar go away for 5-10 minutes to dry the surface going from agar that permitting the bacteria to establish for themselves and on the media. Invert the plate and incubate them at 37^oC for 18-24 hours (Table 1).

In-vivo Anti bacterial activity

The mice were randomly divided into five groups (n=2), for the preparation of inoculating the bacteria on to nutrient agar and incubated at 37^oC overnight. Before inoculation, the mouse models of bacterial skin infection were sedated with either 7. The flanks of the sedated mice were shaved with clippers when necessary and cleansed with an ethanol solution and then apply the bacterial solution on the skin. The treatment with antimicrobial used in this study begins after 4 hours of bacterial inoculation and continued at the regimens of 7 days (Table 2).

In vivo Method – Anti inflammatory activity

Carrageen and induced paw edema in rats

Ten healthy, domestic male albino rats, weighing 230-250 gm were used in this study. These mice were kept in separate cages the room temperature was maintained at 20-25^oC. Carrageenan-induced paw edema model was used for ethanol and aqueous extract of the Lantana camara leaves on inflammation as shown in Table 3. Acute inflammation was produced by the administration of 0.9% mL of carrageenan in the sub plantar region of the left hind paw of a rat. The standard drug (diclofenac) and extract (50mg) were administered 30 min before the carrageenan injection. Inject to the control group and was treated with vehicle alone in the sub plantar region of the left hind paw of rats 8. The standard drug was treated with diclofenac (50mg) used for the evaluation of the anti-inflammatory activity. The volume of the paw was measured immediately and also at the end of 2 hours and 4 hours after the administration of carrageenan using plethysmometer. The percentage inhibition of inflammation was calculated by the following formula:

$\begin{array}{l}\% Inhibition=\\ \frac{Increase in paw volume\left\{\right(control)-(test\left)\right\}}{Increase in paw volume \left(Control\right)}\times 10\end{array}$

In-vivo Method (Wound-healing activity)

The animals were randomly divided into two groups with a sample size of 5 rats/ group. Animals were ketamine (80mg) anesthetized before and during the creation of the wounds 9. A full-thickness excision wound with a circular area of 200 mm and 2 mm depth was created along the markings using toothed forceps, a surgical blade, and pointed scissors. Animals were topical with the simple ointment (Neosporin) base as a placebo control Animals were treated topically with the 10% ointment of the ethanol (200mg/kg daily) and aqueous(200mg/kg daily) extract of Lantana Camara leaves till complete epithelisation as shown in Table 4.

Synthesis Consisting of Silver Nanoparticles on Lantana camara Leaves Squeeze

Ethanolic extract of Lantana camara leaf 10 ml was added to the 90 ml of 1Mm AgNO₃ a ratio of 9:1. This solution was kept at ambient room temperature and stirred continuously for 10 min using a magnetic stirrer 10. After 24 hours changed green colour to dark brown colour which signifies the production of silver nanoparticles.

Characterization of Silver nanoparticles

FT-IR analysis

It turned into performed to spot the functional groups in Lantana Camara that have been liable for the reduction of the silver nitrate and spectracular stabilization of silver nanoparticles 11.

Particle size Analyzer

The basic study that offers information about the share out of the numerous smallest sizes of the particles spread in the stratified sample 12.

Zeta potential

The surface attainable of silver nanoparticles this is fact the depiction of the stability of nanoparticles 13.

RESULTS AND DISCUSSION

Phytochemical Screening

Ethanolic extract and aqueous extract of Lantana camara Leaf extract were screened and the data was given in Table 5.

In-vitro anti-Bacterial Activity

Disc diffusion method

Ethanol extract of Lantana camara, when given for E.coli bacteria, shows a zone of inhibition of 4.5mm, 3.5mm for gentamicin (2mg), ethanol extract of Lantana Camara (2mg) respectively (Figure 1).

Ethanol extract of Lantana camara, when given for Staphylococcus aureus bacteria, shows a zone of inhibition of 6mm, 4.5mm for gentamicin (2mg), ethanol extract of Lantana camara (2mg) respectively (Figure 2)

Zone of inhibition for gentamicin, was slightly more compound to ethanol extract of Lantana camara leaf which is given in Table 6.

From the data of phytochemical screening and in-vitro antibacterial utilisation, it is revealed that the liquid extract of Lantana camara leaves shows more constituents and good anti-bacterial activity compared to ethanol extract.

So it turned into considered so the aqueous extract of leaves as promising extract which was formulated as silver nanoparticles to enhance elegance, acceptability and bio-availability.

Characterization of Silver nanoparticles

UV-Vis spectroscopy

A visible colour change delight in see through to brownness inside of 20 minutes signifies the more formation of AgNPs, which was confirmed by UV-Visible spectrometer (Figure 4). After 70 minutes there has been a vital colour to dark brown due to increased reaction time which reinforces the growth of silver nanoparticles (Figure 3).

The intensity of the absorption peak at 418 nm increased with an increasing period of the aqueous component.

Nanoparticles

FT-IR analysis

The infrared Spectrum used to be analyzing the functional grouping present in Lantana Camara leaf extract (Figure 5, Figure 6). The FTIR spectrums of Lantana camara leaf extract and silver nanoparticles are represented (Table 7).

Particle size Analyzer

Partial size determination of Lantana camara leaf the synthesized AgNPs were shown by the intensity and laser diffraction revealed that particles obtained are a polydisperse mixture with the size ranging from 10 to 30nm. The average diameter of the particles was found to be leaf 2657.2nm (Figure 7).

Zeta potential

The Lantana camara leaf sequenced silver nanoparticles are decided in water as a dispersant. it encounter to leaf 0.3mV (Figure 8).

Plants contain many bioactive chemical constituents with various pharmacological activities. Many potent and effective medicinal properties used for treating fatal diseases have been isolated from various medicinal plants. Hence the demand for medicinal herbal drugs is increasing day by day. The study of the medicinal plants makes miracles in the medical field. Lantana camara was selected for the present study.

The anti-microbial properties of the plant have been examined by numerous researchers worldwide because of the therapeutic potential accredited to Lantana Camara. Collection of Lantana camara leaves and preparation of aqueous and ethanol on the cold extraction method. Further Preliminary phytochemical screening of Lantana camara extracts to identify phytoconstituents. This study can be used in the traditional medicinal system to cure diseases and therapeutic use. The interaction going from aqueous slot with the silver nitrate solution and showed modernized deepening of color into change yellow color to dark brown color.

CONCLUSION

The present research demonstrates that phytochemical constituents of ethanol and aqueous extract of Lantana camara leaves and phytostabilized of AgNPs can act as a unique of antibacterial activity against of organisms like staphylococcus aureus and Escherichia coli responsible for infections in burns and In-vivo methods of Lantana camara leaves extract of antibacterial activity, antiinflammatory activity and wound healing activity. The personalities of water-soluble flavonoids in plant organ extract were responsible for spectracular reduction process of silver nanoparticles. Anti-bacterial activities of ethanol extract of Lantana camara were carried out and it shows that the antibacterial activity of ethanol extract of Lantana Camara is slightly less compared to gentamicin and ceftriaxone. Zone of inhibition for gentamicin, ceftriaxone was slightly more compound to ethanol extract of Lantana camara leaf.

Acknowledgement

The authors are heartily thankful to Dr. C. Appa Rao M. Pharm, Ph. D., Principal, Department of Bio-chemistry, Sri Venkateswara University of pharmaceutical sciences, Tirupati-517501, Andhra Pradesh, India for permitting to do the work and providing all the necessary facilities.

Conflict of interest

The authors attest that they have no conflict of interest in this study.

Funding support

No financial support for the current study.