Formation of Natural Melanin/TiO_(2) Nanostructure Hybrids with Enhanced Optical,Thermal and Magnetic Properties as a Soft Material

The natural Melanin/TiO_(2) was synthesized by the use of ultrasonication under UV radiation.The influence of natural melanin on the structural,optical and thermal properties of TiO_(2) nanoparticles was investigated by using Fourier transform infrared spectroscopy,thermogravimetric analysis and UV-Vis spectroscopy.It was observed that incorporating natural melanin on TiO_(2) nanoparticles(TiO_(2)-Mel)occurred at 2.01 eV with a low value of Urbach energy around 100 meV indicating improvement in the crystalline structure.Magnetic measurement at room temperature showed diamagnetic behavior.Furthermore,thermal results showed that TiO_(2)-Mel is stable even at temperatures up to 400℃.According to the results obtained by the thermal stability of melanin with titanium dioxide,it can be a good candidate in many applications such as solar cells and optoelectronics.

Dietary aflatoxin B1 induces abnormal deposition of melanin in the corium layer of the chicken shank possibly via promoting the expression of melanin synthesis-related genes

San-Huang chicken is a high-quality breed in China with yellow feather, claw and break. However, the abnormal phenomenon of the yellow shank turning into green shank of San-Huang chicken has been a concern, as it seriously reduces the carcass quality and economic benefit of yellow-feathered broilers. In this study, the cause of this abnormal green skin in shank was systematically investigated. Physiological anatomy revealed that the abnormal skin in shank was primarily due to the deposition of melanin under the dermis. After analyzing multiple potential causes such as heredity(pedigree and genetic markers), environment(water quality monitoring) and feed composition(mycotoxin detection), excessive aflatoxin B1(AFB1) in feed was screened, accompanied with a higher L-dihydroxy-phenylalanine(L-DOPA)(P<0.05) and melanin content(P<0.01). So it was speculated that excessive AFB1 might be the main cause of abnormal green skin in shank. Subsequently, the further results showed that a high concentration of AFB1(>170 μg kg–1)indeed induced the abnormal green skin in shank compared to the normal AFB1 content(<10 μg kg–1), and the mRNA levels of TYR, TYRP1, MITE, MC1R and EDN3 genes related to melanin deposition would significantly up-regulate(P<0.01) and the content and activity of tyrosinase(TyR) significantly increased(P<0.05). At the same time, the content of L-DOPA and melanin deposition also increased significantly(P<0.01), which also confirmed the effect of excessive AFB1 on melanin deposition in skin of shank. Results of additional experiments revealed that the AFB1's negative effect on melanin deposition in skin of shank could last for a longer time. Taken together, the results of this study explained the occurrence and possible mechanisms of the abnormal AFB1-related green skin in shank of chickens. Excessive AFB1 in diets increased the L-DOPA content and melanin abnormal deposition in the chicken shank possibly via promoting TyR content and activity, and the expression of melanin synthesis-related genes. Furthermore, our findings once again raised the alarm of the danger of AFB1 in the broiler production.

Pharmacodynamics of frigid zone plant Taxus cuspidata S.et Z.against skin melanin deposition,oxidation,inflammation and allergy

Background:Taxus cuspidata S.et Z.is a precious species of frigid zone plant belonging to the Taxaceae family,which possesses anticancer,anti-inflammatory,hypoglycemic,and antibacterial pharmacological properties.While taxane extracted from Taxus chinensis has been reported to elicit antioxidant activities,whether Taxus cuspidata S.et Z.has skin-protective actions against injuries remained unknown.This study aims to explore the pharmacological effects of three Taxus extracts on skin melanin deposition,oxidation,inflammation,and allergy so as to provide new ideas for the prevention and treatment of various diseases related to skin damage.Methods:Skin melanin deposition was evaluated by measuring melanin content in the skin of guinea pigs by alkali lysis method.Antioxidant capacity was evaluated by measuring superoxide dismutase(SOD)concentration and glutathione(GSH)content in skin tissue homogenates of Kunming mice by SOD assay kit and micro reduced GSH assay kit.The quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting were used to examine the levels of both SOD and recombinant glutathione peroxidase 4(GPX4).Skin inflammation was evaluated by xylene-induced ear swelling test and egg-white-induced paw swelling test in mice.In a mouse model of skin allergy induced by 4-aminopyridine(4-AP),allergy was determined by licking body counts and histamine concentrations in tissue homogenates using enzyme-linked immunosorbent assay(ELISA)kits.Two proinflammatory factors tumor necrosis factor(TNF)-αand interleukin(IL)-1βwere measured by qRT-PCR.Hematoxylin and eosin(HE)staining was conducted to assess the degree of skin lesion.Results:All three Taxus extracts including Taxus chinensis essential oil,Taxus chinensis extract and Taxus chinensis extract compound reduced the melanin deposits in the back skin relative to the non-treated control animals,of which Taxus chinensis essential oil produced the greatest effect.In contrast,the three Taxus extracts elevated SOD and GSH levels in the skin tissues,and the highest increase was seen with Taxus chinensis essential oil.Three Taxus extracts,especially Taxus chinensis essential oil,effectively reduce the rate of ear and paw swelling.All three Taxus extracts reduced the number of body licks,the levels of TNF-αand IL-1β,and the histamine content in tissue homogenates of mice and alleviated skin damage.Consistently,Taxus chinensis essential oil yielded the greatest magnitude of decreases.Conclusion:While all three Taxus extracts possessed the anti-skin melanin deposition,oxidation,and allergy properties,Taxus chinensis essential oil produced the superior effects.

Biomedical overview of melanin.2.Updating molecular modeling,synthesis mechanism,and supramolecular properties regarding melanoma therapy

Melanins represent one of the most ancient and important group of natural macromolecular pigments.They have multiple biological roles in almost all organisms across the Phyla,examples being photoprotection,anti-oxidative action,radical scavenger activity,and heavy metal removal.From the biomedical point of view,melanocytes are involved in the origin of melanoma tumors,and the main therapeutic advances for their treatment have been revised in Part 1 of this review.The chemical structure of eumelanin is a biological concern of great importance,and therefore,exploring theoretical molecular models and synthesis mechanisms will be here described,as well as molecular orbital features and supramolecular organization,which are responsible for the key properties that make these biological pigments so important,and so fascinating.Ultimately,this updated overview is devoted to describe present structural models and physico-chemical characteristics of eumelanin,in order to explain and utilize melanin properties on which new photothermal and ultrasonic protocols for melanoma treatment can be devised and applied.

Oxidative stress regulated heme-oxygenase-1 and glutathione S-transferase-m1 gene expression changes in cell lines exposed to melanins

To investigate the effects of oxidative stress on substantia nigra neuronal degeneration and death in patients with Parkinson＇s disease, we treated neuroblastoma cells （SK-N-SH） and glioma cells with Fenton＇s reagent, iron chelating agent, neuromelanin and dopamine melanin. We investigated the changes in expression of nine oxidative stress-related genes and proteins. The levels of mRNAs for heme-oxygenase-1 and glutathione S-transferase-ml were significantly reduced in SK-N-SH cells exposed to oxidative stress, and increased in glial cells treated with deferoxamine. These results revealed that SK-N-SH neurons react sensitively to oxidative stress, which implies different outcomes between these two types of cells in the substantia nigra. Moreover, the influences of neuromelanin and dopamine melanin on cell function are varied, and dopamine melanin is not a good model for neuromelanin.

Optimization of Conditions for the Extraction of Melanin from Walnut Husk

[Objective] This study was conducted optimize the conditions for the ex- traction of melanin from walnut husk. [Method] Melanin was isolated from walnut husk through alkali extraction and acid precipitation, and the factors that may influ- ence the extraction efficiency： temperature, duration, NaOH concentration and solid- liquid ratio were set at different levels. [Result] The optimal conditions for the ex- traction of melanin from walhut husk were temperature of 80 ℃, extraction duration of 120 min, NaOH concentration of 70 mol/L, and solid-liquid ratio of 1：20. [Conclu- sion] The optimal conditions for the extraction of melanin from walnut husk deter- mined in the present study will provide references for the development and utiliza- tion of natural melanin.

Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment

Objectives: To investigate possible sources of Stenotrophomonas maltophilia(S. maltophilia) in the clinical environment.Methods: Different samples were collected from Amol City of Iran. Steps for the identification of S. maltophilia included culturing, biochemical tests, polymerase chain reaction(PCR) of 16 S r RNA gene and 23 S r RNA gene. In addition, production of melanin pigment and patterns of motility of the bacteria, were also investigated.Results: In our study, 20 S. maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/110(6.36%), blood was 1/777(0.13%),sputum was 4/40(4%), urine was 1/2 947(0.03%), tap water was 1/240(0.42%) and dental suction was 6/120(5%). The isolated bacteria showed production of melanin pigment with rates of strong, moderate, weak, and lack of pigment. Types of motilities were seen in isolates.Conclusions: The highest percentage of bacteria is isolated of oxygen manometer system and dental suction, yet has not been reported from oxygen manometer system. These bacteria have also been associated with patients who have respiratory problems, so it is essential for staffs of hospitals to draw attention to this source of bacteria.

The leaf extract of Mallotus japonicus and its major active constituent, rutin,suppressed on melanin production in murine B16F1 melanoma

Objective: To find anti-melanogenesis materials used in whitening cosmetics.Methods: The ethanolic leaf extract of Mallotus japonicus(M. japonicus) having an anti-melanogenesis activity was separated by a sephadex LH-20 chromatography. Each fraction was measured for its tyrosinase inhibitory activity together with its polyphenol content using the Folin–Ciocalteu method. The anti-melanogenesis activity of the active fractions was determined by the melanin content in the murine B16F1 melanoma. The active fractions were put together due to similar constituents, and then separated by high performance liquid chromatography using a C-18 ODS column. The major antimelanogenesis compound was identified using1 H and13C-NMR and liquid chromatography-mass spectrometry.Results: The ethanolic leaf extract of M. japonicus showed an anti-tyrosinase activity with a high polyphenol content, resulting in suppression of melanin production in the B16F1 melanoma. The extract was separated and the active compound was identical as rutin based on the1 H,13C-NMR and liquid chromatography-mass spectrometry analysis data. In addition, the rutin treatment with cells reduced the melanin content in a concentration dependent manner without any cell toxicity. The leaf extract of M. japonicus containing rutin would be useful in whitening cosmetics for protection from UV-light exposure to be limiting the accumulation of melanin in skin.Conclusions: The leaf extract of M. japonicus and/or rutin isolated from the extract as a key whitening agent would be useful as a whitening cosmetic material for protecting against disorder skin due to melanin accumulation.

Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves.The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor.Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata.Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor(tricyclazole,TCZ),a DNA replication inhibitor(hydroxyurea,HU),and an autophagy inhibitor(3-methyladenine,3-MA),on appressorium turgor and glycerol content.As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.Glycogen and liposomes are the main substances producing glycerol.It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development.This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection.

Therapeutic effect of natural melanin from edible fungus Auricularia auricula on alcohol-induced liver damage in vitro and in vivo

This study explored the therapeutic effects of Auricularia auricula melanin(AAM)on alcoholic liver damage in vitro and in vivo.Human normal liver L02 cells were pre-treated with ethanol and then treated with AAM to explore the therapeutic effect of AAM on ethanol-induced hepatocyte injury.The results show that AAM signifi cantly elevated the cell viability,ameliorated the cell morphology,reduced the ROS and increased the GSH/GSSG of ethanol-pretreated L02 cells.Then,mice were administered with ethanol to induce acute alcoholic liver damage,and administered with AAM to further study the therapeutic effect of AAM on alcoholic liver damage in mice.As a result,AAM reduced the levels of ALT,AST,TG,and MDA,increased the levels of ADH,SOD,and CAT in liver damage mice.The therapeutic effect of AAM may be related to inhibition of CYP2E1 expression and activation of Nrf2 and its downstream antioxidase.The research enriched the bioactivity of AAM and provided some ideas for the development of melanin-related health foods.

Biomedical overview of melanin.1.Updating melanin biology and chemistry,physico-chemical properties,melanoma tumors,and photothermal therapy

Melanins(eumelanin,pheomelanin,and allomelanin)represent a very,if not themost,important group of biological pigments.Their biological roles are multiple,from photoprotection to antioxidant activity,heavy metal disposal or the myriad uses of color in organisms across all Phyla.In the first part of this review,eumelanin biology and some chemical aspects will be presented,as well as key physico-chemical features that make this biological pigment so interesting.The principal characteristics of the melanocyte,the melanin-synthesizing cell in mammals,will also be introduced.Transformed melanocytes are the cause of one of the most devastating known cancers:the malignant melanoma.Epidemiology and molecular signaling aspects will be presented next,as well as the principal advances in promising oncotherapies designed and applied for the treatment of melanoma.In particular,on account of the photo-physical properties of melanin,special details will be provided regarding the use of photothermal therapy for melanoma treatment.

Bacterial melanin promotes recovery after sciatic nerve injury in rats

Bacterial melanin, obtained from the mutant strain of Bacillus Thuringiensis, has been shown to promote recovery after central nervous system injury. It is hypothesized, in this study, that bacterial melanin can promote structural and functional recovery after peripheral nerve injury. Rats subjected to sciatic nerve transection were intramuscularly administered bacterial melanin. The sciatic nerve transected rats that did not receive intramuscular administration of bacterial melanin served as controls. Behavior tests showed that compared to control rats, the time taken for instrumental conditioned reflex recovery was significantly shorter and the ability to keep the balance on the rotating bar was significantly better in bacterial melanin-treated rats. Histomor- phological tests showed that bacterial melanin promoted axon regeneration after sciatic nerve injury. These findings suggest that bacterial melanin exhibits neuroprotective effects on injured sciatic nerve, contributes to limb motor function recovery, and therefore can be used for rehabil- itation treatment of peripheral nerve injury.

Detection of Ca^(2+)-dependent acid phosphatase activity identifies neuronal integrity in damaged rat central nervous system after application of bacterial melanin

The study aims to confirm the neuroregenerative effects of bacterial melanin （BM） on central nervous system injury using a special staining method based on the detection of Ca^2＋-dependent acid phosphatase activity. Twenty-four rats were randomly assigned to undergo either unilateral destruction of sensorimotor cortex （group I; n = 12） or unilateral rubrospinal tract transection at the cervical level （C3-4） （group II; n = 12）. In each group, six rats were randomly selected after surgery to undergo intramuscular injection of BM solution （BM subgroup） and the remaining six rats were intramuscularly in）ected with saline （saline subgroup）. Neurological testing confirmed that BM accelerated the recovery of motor function in rats from both BM and saline subgroups. Two months after surgery, Ca^2＋-dependent acid phosphatase activity detection in combination with Chilingarian＇s calcium adenoside triphosphate method revealed that BM stimulated the sprouting of fibers and dilated the capillaries in the brain and spinal cord. These results suggest that BM can promote the recovery of motor function of rats with central nervous system injury; and detection of Ca^2＋-dependent acid phosphatase activity is a fast and easy method used to study the regeneration-promoting effects of BM on the injured central nervous system.

Comparison of pigment composition and melanin content among white,light-green,dark-green,and purple morphs of sea cucumber,Apostichopus japonicus

Sea cucumber, Apostichopus japonicus（Selenka）, is a commercially important marine species in China. Among the differently colored varieties sold in China, white and purple sea cucumbers have the greatest appeal to consumers. Identification of the pigments that may contribute to the formation of different color morphs of sea cucumbers will provide a scientific basis for improving the cultivability of desirable color morphs. In this study,sea cucumbers were divided into four categories according to their body color： white, light green, dark green, and purple. The pigment composition and contents in the four groups were analyzed by high performance liquid chromatography（HPLC）. The results show that the pigment contents differed significantly among the white, lightgreen, dark-green, and purple sea cucumbers, and there were fewer types of pigments in white sea cucumber than in the other color morphs. The only pigments detected in white sea cucumbers were guanine and pteroic acid.Guanine and pteroic acid are structural colors, and they were also detected in light-green, dark-green, and purple sea cucumbers. Every pigment detected, except for pteroic acid, was present at a higher concentration in purple morphs than in the other color morphs. The biological color pigments melanin, astaxanthin, β-carotene, and lutein were detected in light-green, dark-green, and purple sea cucumbers. While progesterone and lycopene,which are also biological color pigments, were not detected in any of the color morphs. Melanin was the major pigment contributing to body color, and its concentration increased with deepening color of the sea cucumber body. Transmission electron microscopy analyses revealed that white sea cucumbers had the fewest epidermal melanocytes in the body wall, and their melanocytes contained fewer melanosomes as well as non-pigmented pre-melanosomes. Sea cucumbers with deeper body colors contained more melanin granules. In the body wall of dark-green and purple sea cucumbers, melanin granules were secreted out of the cell. The results of this study provide evidence for the main factors responsible for differences in coloration among white, light-green, darkgreen, and purple sea cucumbers, and also provide the foundation for further research on the formation of body color in sea cucumber, A. japonicus.

Melanin change of retinal pigment epithelium and choroid in the convalescent stage of Vogt-Koyanagi-Harada disease

AIM:To observe the melanin change of the retinal pigment epithelium(RPE)and choroid in the convalescent stage of Vogt-Koyanagi-Harada(VKH).METHODS:A retrospective study was performed on 40 eyes of 20 patients in the convalescent stage of VKH.Fundus photography(FP),multi-spectral imaging(MSI),and optical coherence tomography(OCT)were performed.RESULTS:In the VKH convalescent stage,focal RPE melanin accumulation(FRMA)was detected in 34 eyes(85%)on MSI and in 7 eyes(17.5%)on FP.FRMA was limited to the previous retinal detachment area in all 28 eyes(FRMA was detected in 34 eyes on MSI,which were enrolled,and 6 eyes lacked data in the acute stage).Sunset-glow fundus was detected in 20 eyes(50%)on FP.The mean density of FRMA in a 1-mm-diameter circular area of the fovea was 0.04±0.07 on MSI,which was significantly correlated with sunset-glow fundus(ρ=0.467,P=0.02).CONCLUSION:In the VKH convalescent stage,FRMA is derived from the RPE melanin change,and sunset-glow fundus is derived from the choroid melanin change.A higher density of FRMA in the fovea and sunset-glow fundus represents more serious depigmentation of melanin.

Expression of <i>T4HR1</i>, a 1,3,6,8-Tetrahydroxynaphthalene Reductase Gene Involved in Melanin Biosynthesis, Is Enhanced by Near-Ultraviolet Irradiation in <i>Bipolaris oryzae</i>

Bipolaris oryzae is the causal agent of brown spot disease in rice and produces the dark pigment melanin. We isolated and characterized T4HR1 gene encoding 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) reductase, which converted 1,3,6,8-THN to scytalone in the melanin biosynthesis from B. oryzae. A sequence analysis showed that the T4HR1 gene encoded a putative protein of 268 amino acids showing 50% - 99% sequence identity to other fungal 1,3,6,8-THN reductases. Targeted disruption of the T4HR1 gene showed a different phenotype of mycelial color due to an accumulation of shunt products compared to those of wild-type on PDA plates using tricyclazole as a melanin biosynthesis inhibitor. A quantitative real-time PCR analysis showed that the expression of T4HR1 transcripts was enhanced by near-ultraviolet (NUV) irradiation and regulated by transcriptional factor BMR1, similar to three other melanin biosynthesis genes (polyketide synthase gene [PKS1], scytalone dehydratase gene [SCD1], and 1,3,8-THN reductase gene [THR1]) in the melanin biosynthesis of B. oryzae. These results suggested that common transcriptional mechanisms could regulate the enhanced gene expression of these melanin biosynthesis genes by NUV irradiation in B. oryzae.

Bacterial melanin in rat models of Parkinson's disease: a potential neuroprotective strategy

Melanins are widely used in medicine, pharmacology and cosmetics. Different technologies have been used to obtain melanin including： chemical synthesis based on oxidation of tyrosine and its derivatives; extraction from animal materials; alkaline extraction from plant material; and microbiological synthesis. A few number of works have been published that were focused on purification of water insoluble 3,4-dihy- droxy-phenylalanine-melanins （Kukulianskaia et al., 2002）. The majority of synthetic and natural melanins are insoluble in wa- ter that significantly complicates preparation of pharmacolog- ical and cosmetic preparations. Obtaining of low-cost soluble biotechnological melanin can speed up application of melanin in medicine and other fields. For the first time, melanin-syn-thesizing strain with high level of pigment synthesis - Bacillus thuringiensis was obtained. The ecologically safe technology of biosynthesis, isolation and purification of the bacterial melanin has been elaborated.

Isolation and Properties of Melanin in Fine Yak Hair

Melanin granules were isolated from fine yak hair by means of protein enzyme and hydrochloric acid.Resultsof SEM investigation and amino acid analysis were usedto study the physical and chemical properties of melaningranules.The solubility of melanin granules in differentchemical solutions was also studied.

Expression and contribution of microphthalmia-associated transcription factor to the melanin deposition in Liancheng white ducks

The present study investigates the expression of microphthalmia-associated transcription factor(MITF) and its contribution to the melanin deposition in Liancheng white ducks.Nested PCR was used to clone the MITF gene sequence from the skin tissue of female Liancheng white ducks.Ultraviolet spectrophotometry was used to detect the melanin deposition.MITF mRNA expression and melanin deposition in different tissues and organs were detected and their correlation was analyzed.The MITF gene(GenBank number: MG516570) was 1 323 bp in length,contains a complete CDS region(34-1 323 bp) and codes 429 amino acids with 100% homology to the MITF of Anas platyrhynchos and over 95% homology to those of Gallus gallus and Coturnix japonica.Genetic evolution analysis reveals a close relationship of Liancheng white ducks with A.platyrhynchos,and also to lesser extents with Anser cygnoides,silky fowl and G.gallus,as well as Sus scrofa,Ovis aries and other mammals.Real-time quantitative PCR(qPCR) analysis demonstrated that MITF was expressed in skin,gizzard,liver,kidney and muscle,and of these tissues,its expression was the highest in the skin tissue(skin>gizzard>liver>kidney>muscle).Ultraviolet spectrophotometry showed that melanin deposition was positively correlated with the MITF expression level in these five tissues and organs(P<0.05).Together,these results demonstrated a tissue-specific pattern of MITF expression and a positive correlation between MITF expression and melanin deposition,indicating that MITF expression may contribute to the melanin deposition in Liancheng white ducks.

Photothermal Effect-based Cytotoxic Ability of Melanin from Mytilus edulis Shells to Heal Wounds Infected with Drug-resistant Bacteria in vivo

Objective Owing to antibiotic abuse and the subsequent development of antibiotic resistance,bacterial infection has become one of the most persistent unresolved problems.New antibacterial agents,especially those that are environmental-friendly,are urgently needed.Methods Melanin extracted by filtration centrifugation and acid and proteolytic hydrolysis was characterized using UV,FTIR,TEM,and XPS.Photothermal conversion was calculated,and the bacteriostatic effects,in vitro and in vivo,were assessed by plate counting and ratios(%)of wound areas.Results Natural melanin hydrolyzed by trypsin had good photothermal conversion effects,which resulted in superior bacteriostatic activities.The extracted melanin along with laser NIR irradiation at808 nm promoted the healing of wounds infected by drug-resistant bacteria in vivo and was biocompatible according to toxicity tests in vivo and in vitro.Conclusion The present findings indicated a safe and efficient method of developing natural antibacterial agents.