Bio-Fabrication and Efficiency Analysis of Annona Muricata Extract Coated on Bamboo Fiber for Wound Healing Application

ABSTRACT

This study focusses on the bio-fabrication of leaf extracts of Annona muricata dyed bamboo woven fabric and its application in wound healing. Annona muricata was exposed to ultrasonic-assisted extraction (UAE) with acetone for fabric finishing. Bioactivity is characterized and employed in textile fabric coating to evaluate the efficacy of its antibacterial and wound healing characteristics. The treated fabric was then tested for antibacterial activity using gram-positive and gram-negative bacteria, aroma, color intensity, olfaction, and invivo wound healing. The invivo test was carried out in wistar rats for 21 days for 50%, 75%, and 100%. According to the results of the analysis, the extract is a good antibacterial agent against gram-positive bacteria (Bacillus subtilus) and gram-negative bacteria (Pseudomonas sp.), which aids in the prevention of bacteria and other germs from sticking to the injured area since the leaf extracts have high antibacterial properties. The wound healing impact of the plant extract coated with bamboo fabric on in vivo wound models in rats (Wistar albino rats) demonstrated a significant increase in wound closure, showing the efficacy of the coated fabric.

摘要

研究了番荔枝叶提取物对竹编织物的染色及其在伤口愈合中的应用. 用丙酮对番荔枝进行超声波辅助提取（UAE)，用于织物整理. 生物活性物质被表征并用于纺织品涂层，以评估其抗菌和伤口愈合特性的功效.然后使用革兰氏阳性菌和革兰氏阴性菌测试处理过的织物的抗菌活性、香气、颜色强度、嗅觉和内脏伤口愈合. 在 wistar 大鼠中进行了为期21天的invivo试验. 分别为50%、75%和100%. 根据分析结果，该提取物是对革兰氏阳性菌（枯草芽孢杆菌）和革兰氏阴性菌（假单胞菌属）的良好抗菌剂，由于叶提取物具有高抗菌性能，因此有助于防止细菌和其他细菌粘附在损伤区域.用竹织物包裹的植物提取物对大鼠（Wistar白化大鼠）体内伤口模型的伤口愈合影响表明，伤口闭合显著增加，显示了包裹织物的功效.

KEYWORDS:

• Annona muricata
• bamboo fabric
• GCMS
• FTIR
• Olfaction assay
• antimicrobial activity
• antioxidant activity
• wound dressing
• wound healing

关键词:

• 番荔枝
• 竹织物
• 嗅觉测定
• 抗菌活性
• 抗氧化活性
• 伤口敷料
• 伤口愈合

Introduction

The global burden of diseases connected with wounds is becoming a major public health concern. Existing treatments are frequently costly, time consuming, and ineffective in chronic wounds. The problem of overcoming the present wound care hurdles necessitates the use of novel management strategies. Regenerative medicine is a new area of study that focuses on the repair, replacement, or regeneration of cells, tissues, or organs in order to restore the compromised function (Pang et al. 2017).

Wound healing refers to the biological process of tissue growth or the replacement of impaired tissues in the wound area. Skin is the largest organ in the body that is used to cover the entire external surface of our body. It consists of three layers, namely Epidermis, Dermis, and Hypo-dermis. These layers together act as a protective physical barrier to the external factors that affect the skin. When these layers are damaged, the tissue repair mechanism is triggered. Wound healing is of 4 stages: Hemostasis, Inflammation, Proliferation, Maturation (Ellis, Lin, and Tartar 2018).

Price has a great impact on the use of medical care products. Wounds that people suffer from are classified as: Chronic wounds – these types of wound mainly occur in diabetics and in people with high obesity (Martinengo et al. 2019). They stay longer in the body and are considered as a major health issue. They occur mostly in aged people. Pressure ulcer – It is a type of wound that occurs in the epidermis and dermis layers of the skin and the curing process is long, running into months. It increasingly occurs with age and with symptoms such as malnutrition, skin perfusion, etc. Infection – it occurs due to the microorganisms that surround a wound. The major reason for an infection is environmental conditions and the factors that affecting it. Infection can be treated medically (Headlam and Illsley 2020).

Secondary metabolite contribute to the ecological functions (Biswas and Mukherjee 2003). It is found in plants, animals, and in microorganisms. It has made a great impact on pharmaceuticals. Secondary metabolites vary for each plant species. These are divided into three major types – terepenoids, phenolic compounds, and alkaloids. The metabolites mostly take place in low concentrations and depend on the extraction methods (Vijayakumar and Raja 2018).

The wide range of organic molecules that plants create are not directly connected to the main metabolic processes of growth and development. It has only lately been clear how important these natural compounds or secondary metabolites are to the functioning of plants. These metabolites are frequently distinctive to the individual plant species, meaning that every plant species has its own distinct collection of secondary metabolites (Ribeiro de Souza et al. 2009). Leaves are used as a natural remedy to treat spasms, inflammation, hypoglycemia, collapses, and other conditions The plant’s leaf has earned the moniker “the cancer killer,” and as the name implies, it is also employed in conventional medicine to treat cancer. It has various properties in which the chemically active metabolites are source derived from the plant. The plant has been used a good source of medicine. The extracts of these plants have extraordinary secondary metabolite compounds present in them. Such metabolites are alkaloids, phenols, tannins, saponins, etc (Abdul Wahab et al. 2018). But most recent research concentrates on composites reinforced with short bamboo fibers.

A fabric is a group of fibers that has been arranged or pressed together using a variety of methods to create a continuous substance. Bamboo is pyrolyzed in the absence of air to produce bamboo tar and bamboo charcoal; the latter is used as a convenient solid fuel and an efficient adsorbent for removing humidity and smells. Among these, bamboo is recognized as one of the most popular bioresources, and its adsorption characteristics have been the subject of many studies. Chitin, a significant component of the outer skeletons of crustaceans, is the source of the natural biopolymer, chitosan. This substance is well known in the field of wound treatment for its hemostatic qualities (Shanmugasundaram and Gowda 2011).

It is quite difficult to control the stretch and recovery properties of weft-knitted regular rib fabrics. The purpose of this study is to investigate how changes in stitch length, yarn count, and GSM affect the characteristics of weft-knit regular rib cloth for stretch and recovery.

Materials and methods

Plant collection

The leaves of Annona muricata, used for wound dressing, were collected from local fields in Peelamedu, Tamilnadu. They were washed with running tap water and the leaves were pat dried with clean cloth. Finally, the sterilized leaves were shade dried at 37°C for 4–7 days.

Ultrasonic assisted extraction (UAE)

The dried leaves were then ground into a fine powder and suspended in acetone (1:10) to retain the bioactive chemicals found in the leaves. Following this, the powdered samples were treated to ultrasonic-aided extraction with an acetone-water (80:20) solution. Ultrasonic light analyses were carried out using a LABMAN Probe sonicator (model -PRO-650) with a power output of 60 W and a frequency recurrence of 50 KhZ. The tank’s dimensions were 150 × 140 × 100 mm. The necessary chemicals were extracted with this analyzer by rupturing the cell wall with ultrasonic sound waves under optimal conditions (pulse: 5 sec, temperature :-5°C, time: 30 min). The produced extract was next subjected to dark maceration, in which the sonicated extract was held in the dark, overnight, by wrapping it in a foil and storing it at room temperature. The leaf extract was then separated and diluted at different concentrations of 100%, 75%, and 50% (Nolasco-González et al. 2022).

Fabric finishing by bioactive extracts

A laboratory-scaled padder was applied to the extract on a cloth. The pressure between the pads was set to two bars and the rotation speed was set to two meters per minute. The cloth was mixed with extracts of varying concentrations, and the wet fabrics were dried in a hot air oven at 55°C for 20 minutes. The dried and coated materials were then exposed to room temperature for 18 hours (Ramachandran 2004).

Preliminary qualitative analysis

(Sorescu et al. 2018).

Test for tannins (Ferric chloride test)

5% of ferric chloride solution was added to 1 mL extract. The appearance of blue-black indicates the presence of tannins.

Test for saponins (Foam test)

The extract was shaken vigorously with 20 mL of water and observed for persistent foam, which indicates the presence of saponins.

Test for flavonoids (Alkaline reagent test)

Few drops of 10% of NAOH was added to 1 mL of extract. An intense yellow color appeared, which became colorless, on addition of dilute acid, which indicates the presence of flavonoids.

Test for alkaloids (Wagner test)

2 g of iodine and 6 g of potassium iodide were dissolved in 10 mL of distilled water. To this, 1 mL of extract and 1 mlLof prepared reagent were added, which gave a reddish brown color, indicating the presence of alkaloids.

Test for cardiac glycosides (Keller-Killani test)

To the test tubes containing 1 mL of extract, 0.5 mL of glacial acetic acid, 2 drops of 5% ferric chloride and concentrated sulfuric acid were added and observed. The disappearance of reddish-brown color at the junction of the two layers and the bluish green in the upper layer indicates the presence of cardiac glycosides.

Test for terpenoids

To 1 mL of extract, add a few drops of concentrated H₂S0₄. The appearance of reddish brown color indicates the presence of terpenoids.

Test for steroids

5 mL of aqueous plant extract is added to 2 mL of chloroform and concentrated H₂S0₄. The upper layer in the test tube turned red and the H₂S0₄ layer showed yellow with green fluorescence, indicating the presence of steroids.

Thin layer chromatography (TLC)

Thin layer chromatography was used to qualitatively evaluate the phytoconstituents present in the organic extracts (TLC). It was used to characterize the extract from nonvolatile mixtures. In this study, an aluminum foil covered with silica gel on a (60 F254 plate) was employed to identify the bioactive compounds (secondary metabolites) under various mobile phases. TLC was used to analyze the extracts spotted on silica-covered plates for flavonoids, alkaloids, saponins, steroids, tannins-phenols, and lastly, cardio-glycosides. The tannin content was calculated using chloroform, ethyl acetate, and methanol (50:30:20). The saponin and flavonoid concentrations were determined using chloroform: methanol: water (62:36:2) and chloroform: ethanol: glacial acid (94:5:1) (Syarifah, Retnowati, and Soebiantoro 2019). Ethyl acetate: methanol: water was used to examine the alkaloids and cardiac glycosides (81:11:8) Terpenoid components were detected using a mobile phase of toluene: ethyl acetate (93:7) (Kristanti and Tunjung 2015) and steroid components of chloroform: acetone (80:20) (Mariswamy, Gnaraj, and Johnson 2011). The formed plates were then sprayed with vanillin solution (1% (w/v) in 50% phosphoric acid) for steroid detection. The TLC results were further used to validate the presence of tannins, based on the positive reaction (brownish green – blue black coloration) with 0.1% FeCl₃; alkaloids, based on the positive reaction (brown coloration) with Dra-reagent; gendorff’s steroids, based on the positive reactions (violet to blue or green) with acetic anhydride and H₂SO₄; steroidal glycosides, by Keller-Killani test; and cynogenic glycoside, based on the red coloration of the picrate paper. The presence of saponin in the extract was determined by observing the prolonged foaming in distilled water (2 mlL by 1% standard saponin solution (3 mL) (Luciana et al. 2013).

Gas Chromatography and Mass spectrometry Analysis (GC-MS)

The acetonic extract of Annona muricata is used in gas chromatography. The extract was investigated using Turbo Mass ver 5.4.2 software and GC-MS Perkin Elmer Model: clarus 680, which is strengthened with mass spectrometer Clarus 600 (EI). Elite-5 MS (5% biphenyl 95% dimethylpolysiloxane, 30 m 0.25 mm ID 250 m df) wrapped-fused silica column. To separate the components, helium was used as a carrier gas at a constant flow rate of 1 mL/min. When the chromatography began, the injector temperature was set to 260° C. After injecting 1 L of sample into the equipment, the oven was heated to 60° C for approximately 2 minutes, followed by 300° C at a rate of 10° C min⁻¹, and 300° C for roughly 6 minutes. The mass detector was run with the following parameters: scan time 0.2 seconds, scan interval 0.1 seconds, ionization mode electron impact at 70 eV, transfer line temperature 230° C, ion source temperature 230° C, and ionization mode electron impact at 70 eV, scan time 0.2 sec, and scan interval 0.1 sec. The fragments range from 40 to 600 Da. The component spectra was compared to a database of known components recorded in the GC-MS NIST (2008) library. The major chemicals discovered had a retention time of 1.59 (Ezhilan and Neelamegam 2012).

Fourier-transform infrared spectroscopy (FTIR)

The bio-extract powder of Annona muricata was analyzed using Fourier-transform infrared spectroscopy to confirm secondary structures. The samples were taken and mixed with KBr (1:1) before being pelletized using a KBr hydraulic press with a pressure of roughly 10 tons. At the IR range of 500-4000 cm-1, FTIR was utilized to examine the functional portions of the pellet (Younis et al. 2021).

Determination of antioxidant activity using ferric reduction/antioxidant power method

The extracts’ reducing power was measured using the Ferric reducing antioxidant power test (FRAP). FRAP was performed using five different concentrations of Annona muricata extracts (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) and L-ascorbic acid, which was then combined with 2 mL phosphate buffer (0.2 M, pH 6.6) and 2 mL of 1% potassium ferricyanide (K3Fe (CN)6). The mixture was then incubated for 20 minutes at 50° C. Next, 2 mL of 10% trichloroacetic acid (TCA) was added, and the mixture was centrifuged for 5 minutes at 3000 rpm (revolutions per minute). The supernatant (2 mL) was collected and mixed with 0.1% ferric chloride (FeCl3) and 2 mL distilled water. The experiment was carried out in triplicate in each case. The absorbance was then spectrophotometrically measured at 700 nm with a UV-vis spectrophotometer and observed (Guchu et al. 2020).

Treatment of bamboo fabric with Chitosan

Chitosan was used to treat the fabric at 5%. The ratio of material to liquor was 1:30. Chitosan was dissolved in water and held at a temperature of 70° C in a water bath. The fabric was treated for approximately one hour before being dried in a hot air oven (Massella et al. 2019).

Antimicrobial finish application of Bamboo fabric

The fabrics were soaked in 50%, 75%, and 100% acetonic extract of Annona muricata for 30 minutes before being padded separately and compressed to achieve a wet pick up of 85% of the weight of the cloth. The fabric was dried at 80° C for 3 minutes before being restored at 95° C for 10 minutes in a lab model curing chamber (Ramachandran 2004).

Antimicrobial activity assessment of Dyed Bamboo fabric

The nutrient agar plate method was used to conduct antimicrobial testing on the bamboo fabric treated with Annona muricata. The nutrient agar media was created in a petri plate. For antibacterial action, gram-negative (Pseudomonas sp.) and gram-positive (Bacillus subtilus) bacteria were utilized. The prepared nutrient agar plate was inoculated with the aforesaid bacterial culture, using an L-rod approach, and the plates were maintained at 37° C in an incubator for 24 hours. After 24 hours, the antibacterial activity was assessed qualitatively using the Agar diffusion method (ISO 20,645). The dyed Annona muricata fabric and the undyed control fabric samples were placed in close contact with an AATCC bacteriostatic nutrient agar medium plate, and the plates were re-incubated at 37 C for 18–24 hours. After re-incubation, the zone of inhibition study was performed and quantified to determine whether the antibacterial efficiency of the Annona muricata-treated fabric had been compromised. The zone creation indicates the capability of Annona muricata’s antibacterial effect on bamboo fabric (Kamboj, Jose, and Singh 2022).

Determination of color strength

Annona muricata-treated samples were examined in a reflectance spectrophotometer (Gretagmacbeth) with D65 light using Colour-i-control software connected to an IBM PC. Using the Chroma flash Color Matching System, the K/S value for color comparison between A Annona muricata solution-treated cotton samples and the washed samples treated with different cycles was calculated (ASHCO Industries Ltd. India). Kubelka-Munk Equation 1 is used to calculate the color value K/S

(1) $\frac{\mathrm{K}}{\mathrm{S}}=\frac{{\left(1-R\right)}^2}{2R}\times 100$(1)

where,

K shows the absorption coefficient,

S shows the scattering coefficient,

R shows the spectral reflectance of the colored samples at a wavelength of maximum absorption (where the reflection is minimum).

The color fastness to washing test was performed in accordance with ISO 105-C06 (ISO 105 C06 A2S) using a solution of 5 gpl of soap in which the free alkali calculated as Na₂CO₃ should not exceed 0.3%, free alkali calculated as NaOH should not exceed 0.1%, and total fatty matter should not exceed 85%. Fabric samples were treated for 30 minutes at 40 + 2° C. The test was carried out on the gyrowash machine. Following a 10-minute rinse in cold running tap water, the treated samples were rinsed in cold distilled water. After squeezing, the fabric was air dried at a temperature no higher than 60° C.

Fragrance intensity test

The life of a fragrance is determined by the concentration of the aroma and is entirely dependent on the fragrant chemicals. The fragrance intensity of samples treated with Annona muricata was evaluated using a subjective test method in which judges rated the fragrance intensity on a 5-point Likert scale, where 4 represents excellent, 3 represents good, 2 represents fair, 1 represents poor, and 0 represents no fragrance intensity. The experiment was conducted over a period of four weeks (Kert et al. 2021).

Olfaction China GB test

This technique was carried out as soon as the test samples were opened. The experiment was carried out in an area free of unusual odors. After washing his hands and putting on gloves, the test performer held the fabric specimen close to his nostril and sniffed the aroma in the cloth. Meanwhile, the aromatic hydrocarbons were tracked down, and the unusual scent was discovered. Two people carried out the process, and the results are analyzed using the current standard code GB 18,401–2010, which stands for National general safety technical code for textile products (ChineseStandard.net 2010)(stand (Gyesi, Opoku, and Borquaye 2019).

Experimental protocol for in vivo study conducted in wounded rats to validate the efficiency of the bio extract-coated fabrics

The animals were divided into seven groups of five. On Day 0, the animals were anaesthetized, the dorsum of each animal was shaved, and a wound area was formed 5 mm distant from the ears using a circular-colored rubber stamp. The full thickness skin was removed from the delineated region, including the panniculus carnosus, resulting in a wound area of approximately 400 mm². Following wound formation, the experimental animals were separated into seven groups of five animals each. Except for Group I, all groups got the prescribed treatment until the incision was completely closed. On days 0, 7, and 14, their body weights were measured. The wound area was assessed on each alternate day of the trial, namely on days 0, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. The wound area was calculated as a percentage. Table 1 shows the study’s design. Individual animal body weights were measured on days 0, 7, 14 (Moghadamtousi et al. 2015).

Table 1. Experimental Animal design for wound healing analysis in Wistar rats.

S.no	Groups	No. of Animals
I.	Control	5
II.	AM1122–100%	5
III.	AM1122–75%	5
IV.	AM1122–50%	5
Total No. of animals		20

Assessment of wound healing

The physical characteristics of wound healing, specifically wound closure, were investigated by tracing the raw wound. Every other day, the wound area was measured by retracing the wound. The extent of wound healing was determined.

$\mathrm{\%}wound\hspace{0.17em}closure=\frac{\mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{y}0-\mathrm{w}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{y}\mathrm{N}}{wound\hspace{0.17em}area\hspace{0.17em}on\hspace{0.17em}Day\hspace{0.17em}0\hspace{0.17em}}X\hspace{0.17em}100$

Where N= wound area on the corresponding days.

Histopathological analysis

At the end of the trial, the wound tissue from each group was collected and fixed in 10% formalin. After removing lipid debris with alcohol and dehydrating the sections in increasing ethanol concentrations, the tissue samples were embedded in paraffin wax. A 5-10 m-thin coronal piece of wound tissue, with a thickness of 2 mm, was obtained with a microtome. Haematoxylin and Eosin were used to stain the slides (H&E). To measure the changes caused by the different treatments, the stained tissue sections were examined under a motic digital microscope (40×) and the wound tissue region was photographed.

Statistical analysis

One-way analysis of variance (ANOVA) was used to determine the significant intergroup difference of each parameter. Following a significant ANOVA result, Dunnet’s test was employed for individual comparisons. Differences with p 0.05 were deemed statistically significant. For statistical analysis, the GraphPad prism 8 software (GraphPad Software, Inc., California, USA) was utilized.

Results and discussion

Plant secondary metabolites are an especially rich source of pharmaceuticals, food additives, and fine chemicals. They also provide unique materials that are used in a variety of fields. Secondary metabolites are classified according to their chemical make-up (for example, whether they have rings or contain a sugar), composition (whether they contain nitrogen or not), solubility in various solvents, or the process by which they are formed (for example, tannins are produced by phenylpropanoid). Furthermore, they are usually classified according to their metabolic processes. Most people focus on these three large molecular families: Phytochemicals, Terpenes, and Steroids. Alkaloids and Flavonoids are included in Table 2 (Sorescu et al. 2018). Annona muricata was extracted using the ultrasonic-assisted method. Acetone, a polar dissolvable solvent, was used in the extraction process. Ultrasound extraction technique not only can be applied to improve the quality, cost, efficiency and safety of products, but offer a potential value for extracting bioactive compounds from medicinal plants with unique functionality as well. It increases the mass transfer process and avoids the destruction of the active ingredient at high temperature, effectively. Ultrasonic waves stimulates glands by causing relatively minor ultrasonic pressures on plant cells, causing the active substance to be released more quickly. This method is frequently utilized because it can reduce the consumption of organic solvents while increasing the purity of bioactive substances. As a result, the preceding investigations have proved that the UAE technology fits the demand for greener products, sustainable energy, and environmental protection, while also playing a significant role in the protection of secondary metabolites in Annona muricata.

Table 2. Phytochemical analysis of Annona muricata.

Bioactive compounds	Response
Tannins	+
Saponins	-
Flavonoids	-
Cardiac Glycosides	-
Terpenoids	+
Steroids	+
Alkaloids	+

Thin-layer chromatography (TLC) detection of Annonannona muricata

Thin-Layer Chromatography is used to identify the bioactive compounds present in the extracts of Annona muricata as given in Table 3. TLC examination of secondary metabolites reveals the presence of spots, with Rf values at 0.55, 0.552, 0.704, 0.649, 0.846, and 0.554 in the ethanolic extract, and for acetonic extracts, 0.543 and 0.72 was determined (Figure 1).

Figure 1. Thin-layer chromatography analysis of Annona muricata.

Table 3. TLC analysis of Annona muricata.

Phytochemical constituents	Solvent system for TLC	Acetonic extract (Rƒ and color spot)	Ethanolic extract(Rƒ and color spot)
Saponin	Chloroform:Methanol:water (64:32:2)	.72 (blue) (blue)	.704 (blue) (blue)
Tannins	Chloroform:Ethyl acetate:Methanol (50:30:20)	-	.649 (yellow) (yellow)
Steroids	Chloroform:Acetone (80:20)	-	.552 (yellow) (yellow)
Glycosides/Alkaloids	Ethyl acetate:Methanol:Water (81:11:18)	.543 (blue) (blue)	.554 (blue) (blue)
Terpenoids	Toluene:Ethyl acetate (93:7)	-	.55 (dark green) (dark green)
Flavonoids	Chloroform:Ethanol:Glacial acetic acid (94:4:1)	-	.846 (yellow) (yellow)

The figure depicts visualization in UV and non-UV light, which also reveals that ethanolic and acetonic extracts all contain spots that are very similar to one another. As previously mentioned, the test findings of the acetonic extracts of Annona muricata phytochemistry for terpenoids, steroids, saponins, tannins, flavonoids, and alkaloids/glycosides were all positive. This agrees with the presence of secondary metabolites’ in TLC analyses. A lower polarity of the chemical components is indicated by a higher retention factor by the stronger interaction with the plate (Qorina et al. 2019).

Gas chromatography mass spectroscopy analysis of Annona muricata

The objective of this study was to utilize gas chromatography and mass spectroscopy to identify bioactive components in an acetonic extract of Annona muricata leaves (GC-MS), given in Table 4. The GCMS analysis of the acetonic leaf extract was performed according to standard methodology utilizing Perkin-Elmer Gas Chromatography-Mass Spectrometry equipment, and the mass spectra of the chemicals discovered in the extract were matched with the National Institute of Standards and Technology (NIST) library. The GC-MS analysis revealed the presence of chemicals given in Figure 2 such as 5-Amino-3-azido-1,2,4-Triazine-6carbonitrile, Cyclohexanol,4-methyl,trans compound is responsible for anti-oxidant characteristics and also possesses antibacterial activity (Bhardwaj et al. 2022),Tetrahydrofuran-2-one,5-[1-hydroxyhexyl] is responsible for anti-inflammatory behavior and also possesses antidiabetic agents activity (Manikandaselvi and Brindha 2014),Methyl-4-o-methyl.alpha.D-glucopyranoside compound possesses anti-microorganisms capability (Lai et al. 2018). These compounds showed increased biological and pharmacological activities.

Figure 2. GC-MS chromatogram analysis of Annona muricata.

Table 4. GC-Mass spectrum analysis for molecular weight analysis of Annona muricata.

S No.	Retention time	Molecule Name	Molecular Formula	Molecular Weight (g/mol)	Molecular Structure
1.	1.279	5-AMINO-3-AZIDO-1,2,4-TRIAZINE-6-CARBONITRILE	C₄H₂N₈	162.11
2.	1.324	CYCLOHEXANOL,4-METHYL-, TRANS-	C₇H₁₄O	114
3.	1.359	TETRAHYDROFURAN-2-ONE,5-[1-HYDROXYHEXYL]-	C₁₀H₁₈O₃	186
4.	1.594	METHYL-4-O-METHYL.ALPHA. D-GLUCOPYRANOSIDE	C₈H₁₆O₆	208

FTIR analysis of bioactives of Annona muricata

The functional group analysis by IR spectrum was carried out in a fixed cell with an appropriate solvent. Since the ultrasonic-assisted extracted mixture was acetonic extract, the FTIR was performed accordingly. The fixed cell size was about 0.1 mm thickn and a spacer of 0.025 was used in the detection. FTIR characterization of Annona muricata exhibited aromatic rings/functional groups, indicating the aroma flavor. Specific regions of the FTIR spectra (3301.54 cm − 526.01 cm) are observed and are given in Table 5. In the region between 3500–3100 cm⁻¹, the prominent band centered around 3301.54, which is designated to N-H stretching vibration of glycosidic structures. The range between 3000–2850 cm⁻¹ is designated to C-H stretching vibration of alkanes and alkenes. The bands between1800–1500 cm⁻¹ are in turn designated to C=O, C=C stretching vibrations of aldehydes and alkenes; these compounds have the potential to cure various human health problems (Kabila, Sidhu, and Ahluwalia 2020). The range 1373.56 is designated as S=O stretching vibrations of sulfones, sulfonyl chlorides, sulfates, sulfonamides etc. The range between 1300–1000 is designated to C-O stretching vibrations of alcohols, ethers, esters, carboxylic acids, anhydrides etc. In the region between 950–750 cm, two major bands C-X and C-H (aromatic) are assigned to chloride and aromatic proteins. The final region of 526.01 cm⁻¹ is indicated as C-H and C-H (Bromide/Iodide) in the spectrum, given in the Figure 3. Bromide/Iodine are the compounds that havebeen used as disinfectants, antiseptic agents, and also for the treatment of wounds (Selvaggi et al. 2003).

Figure 3. Fourier transform infrared spectrum analysis of Annona muricata.

Table 5. FTIR spectrum for Annona muricata.

Peak frequency range (cm^−1)	Functional group
3301.54 (cm^−1)	Primary and secondary amines & amides (N-H) (N-H)
292.53 (cm^−1)	Alkanes (C-H)
2821.64 (cm^−1)	Aldehyde (C-H)
1731.78 (cm^−1)	Aldehyde (C=O)
1613.39(cm^−1)	Alkene (C=C)
1514.34(cm^−1)	Nitro compound(N-O)
1373.56(cm^−1)	Sulfones, sulfonyl chlorides, sulfates, sulfonamides (S=O)
1235.04(cm^−1)	Alcohols, Ethers, Esters, Carboxylic acids, Anhydrides (C-O)
1032.81(cm^−1)	Alcohols, Ethers, Esters, Carboxylic acids, Anhydrides(C-O)
526.01(cm^−1)	Bromide, Iodide (C-X)

Determination of antioxidant activity using ferric reduction/Antioxidant power method

At a wavelength of 700 nm, the acetonic extract of Annona muricata showed notable concentration-dependent increases in absorbance values. To determine the reducing activities, different concentrations of acetonic extracts (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/mL) and L-ascorbic acid at the same quantities were used as a standard. The extracts of Annona muricata had shown considerably higher absorbance values at all of the tested doses. The reduction of ferric ions was higher in the acetonic extract of Annona muricata at a concentration of 90 µL as shown in Figure 4.

Figure 4. Graphical representation of antioxidant activity expressed by Annona muricata.

Antibacterial activity assessment of dyed bamboo fabrics with Annona muricata extract

Antibacterial testing was performed on fabric samples treated with Annona muricata leaf extract using gram-positive bacteria (Bacillus subtilis) and gram-negative bacteria (Pseudomonas sp.). The zones of inhibition of distinct samples examined with extracts of 50% (Sample A), 75% (Sample B), and 100% (Sample C) concentrations are depicted in the Figure 5a. For 50%, 75%, and 100% extracts, zones of inhibition of 9 mm, 11 mm, and 10.6 mm were detected.

Figure 5a. Zone of inhibition against gram-positive bacteria from the leaves of Annona muricata.

Figure 5b. Zone of inhibition against gram-negative bacteria from the leaves of Annona muricata.

Antibacterial testing was also performed on the treated samples using gram-positive bacteria (Bacillus subtilus). The zones of inhibition of several samples examined with extracts of 50% (Sample A), 75% (Sample B), and 100% (Sample C) concentrations are depicted in the Figure 5b. For 50%, 75%, and 100% extracts, zones of inhibition of 23.6 mm, 24.3 mm, and 26.3 mm were found.

(Tables 6 and 7) depicts the differences in the zone of inhibition observed between gram-negative and gram-positive bacteria and indicated in Figure 6. Gram-positive and gram-negative bacteria have different cell wall compositions. The cell walls of gram-positive and gram-negative bacteria differ structurally (Vijayameena et al. 2013).

Figure 6. Graphical representations of the zones of inhibition against gram-positive and gram-negative bacteria.

Table 6. Zones of incubation measured for different concentration of Annona muricata against Bacillus subtilis.

Gram positive bacteria: - Bacillus subtilis
Extract concentration (%)	Zone of incubation (diameter (mm))
50%	23.6 mm
75%	24.3 mm
100%	26.3 mm

Table 7. Zones of clearance measured for different concentration of Annona muricata against Pseudomonas sp.

Gram negative bacteria: - Pseudomonas
Extract concentration (%)	Zone of incubation (diameter (mm))
50%	9 mm
75%	11 mm
100%	10.6 mm

Computer color matching system (CCMS)

The spectral reflectance of untreated and Annona muricata extract-treated fabrics was measured and shows that the K/S value of bio extract increases as the concentration of the extracted solution increases. It shows that the depth of shade relies on the content of the leaf extract-treated samples. The k/s value of an untreated fabric is 12 (Ramaiah and Ari 2019) and the treated fabric value is identified under CCMS color matching system as 14.

Fragrance intensity testing

A fragrance intensity test was performed in accordance with the subjective approach. Even without washing, the smell intensity in the fabric gradually deteriorated with time in natural environmental settings. Utilizing the subjective technique, the rating of fragrance strength was determined to be 3 at the conclusion of the fourth week, on a Likert scale of 0 (no aroma) to 4 (greater aroma intensity). The bar chart Figure 7 shows a graphical representation of the fragrance strength, with the 100% extract-treated fabric having a higher fragrance rating than the 75% and 50% concentrations. As a result of its superior binding property, 100% treated cloth keeps the aroma better than 75% and 50% treated fabric. According to subjective studies, this smell can endure up to 20 washes and for more than 4 weeks under natural ambient settings. This enables the apparel and textile industries to use fragrance-treated fabric in the development of new and hygienic apparel goods in the future (Kert et al. 2021).

Figure 7. Fragrance Intensity.

Olfaction China GB test

The olfaction method was used to determine the distinctive odor of Annona muricata-treated fabric; this is given in Table 8 (Wu et al. 2019). According to the results of the analysis, our fabric meets the GB/T 18,885–2009 [Technical Specification of Ecological Textiles code] and GB/T 20,097–2006 [Protective Clothing code] standards (ChineseStandard.net 2010). This study shows that the leaves of Annona muricata offer a good odor (Gyesi, Opoku, and Borquaye 2019).

Table 8. Olfaction GB Test analysis of the treated fabric.

Test Performer	Type of smell	Herbal smell	Aromatic smell	Good/bad odor
Undyed fabric	No fragrance	No	No	No
50% dyed fabric	Peculiar	Yes	Yes	Good
75% dyed fabric	Peculiar	Yes	Yes	Good
100% dyed fabric	Peculiar	Yes	Yes	Strong

Wound closure in wistar rats treated with Annona muricata wound dressing material

Effect of body weight

The body weight data were tracked once every 7 days throughout the trial, as indicated in Table 9. On day one, the body weights of all the groups with various treatment animals were measured and recorded. There is no significant difference in body weight seen and reported from day 7 to day 21, shown in Figure 8.

Figure 8. Effect of bioextract formulation of Annona muricata on body weight of treated and untreated wistar rats (gms).

Table 9. Effect of bioextract formulation of Annona muricata on body weight of treated and untreated wistar rats (gms).

Groups	Treatment	Body weight (gms)
		0 day	1^st day	7^th day	14^th day
I	Control	195 ± 0.21	198 ± 0.18	209 ± 0.87	217 ± 0.84
II	AM1122–100%	194 ± 0.39	198 ± 0.54	210 ± 0.64	218 ± 0.54
III	AM1122–75%	195 ± 0.36	197 ± 0.64	208 ± 0.53	217 ± 0.72
IV	AM1122–50%	194 ± 0.34	198 ± 0.53	209 ± 0.62	218 ± 0.74

NOTE: Values are Mean± SD, n = 5; *p <.05 vs Group I (control), ANOVA followed by Dunnett’s multiple comparison test.

Assessment of wound healing

The wound healing outcomes were documented on alternating days throughout the trial Figure 9, as indicated in Table 10. During the 5th-21st days, there was a significant variation in wound area in the Bio extract-formulation of AM1122 at three different concentrations of 100%, 75%, and 50%, which was observed and recorded, whereas the % wound closure in the 5-day and 9-day period is indicated in Table 11 and a pictorial representation is shown in Figure 10. Bio extract-formulation treated with Annona muricata (AM1122) groups showed a higher significance difference when compared to the control group, as shown in Figure 11. Similar study conducted using various nano particles revealed that the particle size of nanoemulsion plays an important role the therapeutic efficacy of the product. In general, a smaller particle size is beneficial as it can reduce the irritation and enhance the percutaneous absorption (Nastiti et al. 2017). The average particle size of NE5 was found to be the lowest among the tested nanoemulsions; hence, it is incorporated in gel formulations. The zeta potential of vesicles is also an important parameter that describes the physical stability of the colloids (Shah et al. 2021).

Figure 9. Effect of bioextract formulation of Annona muricata on wound area of treated and untreated wistar rats.

Figure 10. Wound area closure analysis of wistar rats, on treatment with Annona muricata bio extract.

Figure 11. Wound closure rates on Wistar albino rats with the dyed bamboo fabric as wound dressing material.

Table 10. Effect of bio extract formulation of Annona muricata on wound area of treated and untreated wistar rats.

Days	Wound area (mm^2)
	G-I (Normal control)	G-II (AM1122–100%)	G-III (AM1122–75%)	G-IV (AM1122–50%)
0	400 ± 0	400 ± 0	400 ± 0	400 ± 0
1	374 ± 2.92	373.6 ± 2.07	374.4 ± 3.29	374.4 ± 2.07
3	288.6 ± 1.14	263.8 ± 1.92	258.6 ± 3.36	266.4 ± 3.85
5	265.6 ± 2.07	146.6 ± 1.95	165.2 ± 1.64	235.4 ± 3.51
7	225.4 ± 2.41	84.8 ± 1.48	96.2 ± 2.77	135.2 ± 3.19
9	172.6 ± 2.7	67.6 ± 2.41	73.6 ± 3.05	98.8 ± 3.35
11	138.2 ± 1.48	27.4 ± 1.14	44.2 ± 2.28	76.4 ± 2.41
13	77.2 ± 2.17	10 ± 1.58	20.2 ± 1.3	45.2 ± 2.39
15	53.2 ± 2.17	1.4 ± 0.55	1.2 ± 0.45	16.2 ± 2.77
17	27.2 ± 1.92	0 ± 0	0 ± 0	1.2 ± 0.45
19	11.4 ± 1.14	0 ± 0	0 ± 0	0 ± 0
21	2.6 ± 0.89	0 ± 0	0 ± 0	0 ± 0

Values are Mean± SD, n = 5; *p <.05 vs Group I (control), ANOVA followed by Dunnett’s multiple comparison test.

Table 11. Wound area closure analysis of wistar rats on treatment with Annona muricata bio extract.

Days	Wound closure (mm^2)
	G-I (Normal control)	G-II (AM1122–100%)	G-III (AM122–75%)	G-IV (AM1122–50%)
0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
1	6.5 ± 0.73	6.6 ± 0.52	6.4 ± 0.82	6.4 ± 0.52
3	27.85 ± 0.29	34.05 ± 0.48	35.35 ± 0.84	33.4 ± 0.96
5	33.6 ± 0.52	63.35 ± 0.49	58.7 ± 0.41	41.15 ± 0.88
7	43.65 ± 0.6	78.8 ± 0.37	75.95 ± 0.69	66.2 ± 0.8
9	56.85 ± 0.68	83.1 ± 0.6	81.6 ± 0.76	75.3 ± 0.84
11	65.45 ± 0.37	93.15 ± 0.29	88.95 ± 0.57	80.9 ± 0.6
13	80.7 ± 0.54	97.5 ± 0.4	94.95 ± 0.33	88.7 ± 0.6
15	86.7 ± 0.54	99.65 ± 0.14	99.7 ± 0.11	95.95 ± 0.69
17	93.2 ± 0.48	100 ± 0	100 ± 0	99.7 ± 0.11
19	97.15 ± 0.29	100 ± 0	100 ± 0	100 ± 0
21	99.35 ± 0.22	100 ± 0	100 ± 0	100 ± 0

Note: Values are Mean± SD, n = 5; *p <.05 vs Group I (control), ANOVA followed by Dunnett’s multiple comparison test.

Histopathological study

The histopathological features of the tissue of all group animals were examined under a motic microscope (10×). From Figure 12, Group-I (Control) animals showed inflammatory cells, reduced collagen fibers, and in the blood vessels, there is also a presence of visible scar tissue. Group-II(100%), III(75%), and IV(50%) which was evident by increased collagen fibers and blood vessel it showed a higher significance when compared to the control group-I. The above results revealed that among all groups, the groups treated with AM1122 showed a higher significance in %wound closure, according to a dose-dependent manner, and a decrease in surface area of wound, which were further observed in histopathology, compared to group I Normal control. The ability of P. americana to repair and reverse the already injured tissues of alloxan-induced rats allows for the observation of its tissue-protective function, and the observed impact is consistent with other researchers’ findings (Adewole and Ojewole 2007). This study reassured the antioxidant properties and the possibility of use in the pharmaceutical and cosmetic industries (Gyesi, Opoku, and Borquaye 2019). Percutaneous absorption is generally influenced by various factors, including the characteristics of drugs, additives, formulation type as well as the skin integrity and transport route (Nair et al. 2013). The potential of vesicular emulgel to transport insulin across the rat membrane was demonstrated recently (Shehata et al. 2020).

Figure 12. Effect of Annona muricata leaves extract on histopathological evaluation.

G I – Control

G II – 100% treated fabric

G III – 75% treated fabric

G IV – 50% treated fabric

Conclusion

Annona muricata was studied using spectral reflectance, GCMS, and FTIR, as well as an antibiotic assay and aroma intensity analysis. The Agar Well Diffusion method is used to investigate antibacterial activity against gram-negative bacteria (Pseudomonas sp.) and gram-positive bacteria (Bacillus subtilus) using textiles treated with acetonic extract solution. The zone of inhibition was measured for various treated fabrics, and revealed that the extracts were a good antibacterial agent against gram-positive bacteria (Bacillus subtilus) and gram-negative bacteria (Pseudomonas sp.); they helped prevent bacteria and other microorganisms from attaching to the fabric surface. As a result, healthcare experts are protected by functional clothes as well as materials used throughout the home, such as socks, mattresses, baby diapers, and coverings. According to the olfaction China GB technique, the treated fabric had a peculiar scent with aromatic and herbal flavors and nice odor. As a result of the superior binding property of Annona muricata-treated cloth, the smell is retained. According to subjective evaluations, this smell can last up to 20 washes and for more than 4 weeks. Besides, pre clinical evaluation indicated the wound healing efficiency of the fabric in treated wistar rats. This enables the clothing and textile industries to use fragrance-treated fabric in the development of new and hygienic clothing goods in the future.

Highlights          

• The leaf extracts of Acalypha indica and Tectona grandis were made into consortia with different concentrations (50% 75%, and 100%) and coated on bamboo woven fiber material for wound dressing applications.

• The bamboo woven fabric was utilized as a textile substrate for this research. Chitosan is used as a cross-linking agent and potassium alum was used as a mordant, and engrossed with various concentrations of the bioactive extract.

• This treated fabric was further evaluated for antimicrobial assay using gram-positive and gram-negative bacteria, fragrance test, color intensity, and olfaction test, followed by in vivo wound healing assay.

• It is asserted that natural extracts coated on bamboo-based dressing materials offer beneficial properties for wound healing and allows the attire and textile industry to utilize bio-extract-treated fabric in developing innovative and hygienic attire products in the future.

Acknowledgements

This work is part of final year B.Tech Biotechnology project of first and second authors. The authors gratefully acknowledge the Department of Biotechnology and Sri Shakthi Institute of Engineering and Technology for providing an ambient environment for the successful completion of the project.

Disclosure statement

No potential conflict of interest was reported by the author(s).