Promising role of Lawsonia inermis L. leaves extract and its nano-formulation as double treatment against aflatoxin toxicity in ulcerated-rats: Application in milk beverage

Dina Mostafa Mohammed; Marwa M El-Said; Ahmed Noah Badr; Doha H Abou Baker; Amal S Hathout; Bassem A Sabry

doi:10.1016/j.heliyon.2023.e19290

. 2023 Aug 28;9(9):e19290. doi: 10.1016/j.heliyon.2023.e19290

Promising role of Lawsonia inermis L. leaves extract and its nano-formulation as double treatment against aflatoxin toxicity in ulcerated-rats: Application in milk beverage

Dina Mostafa Mohammed ^a, Marwa M El-Said ^b, Ahmed Noah Badr ^c, Doha H Abou Baker ^d, Amal S Hathout ^c, Bassem A Sabry ^c,^∗

PMCID: PMC10480603 PMID: 37681189

Abstract

Aflatoxins are an unavoidable contaminant of foods. The current work aimed to study the ameliorating effect of Lawsonia inermis L. extract and its nano-formulation versus aflatoxin ingestion in ulcerative rats. Lawsonia inermis L. bioactivity was evaluated by both antioxidant & antimicrobial assays. The nanoparticles characterization measurements were evaluated. Different parameters in the fortified milk beverage were assessed. Seventy two Sprague-Dawley male rats were randomized into 12 groups (6 rats/group) where peptic ulcer was induced with a single aspirin dose (500 mg/kg BW) orally. The nutritional and biochemical parameters were evaluated. The results showed that antioxidant activity and total phenolic content increased with increasing nano-formulation ratio. A remarkable improvements in all the treated groups, either for ulcer alone or for aflatoxin exposed ulcerative groups in normal and nano-formulation. Conclusively, Lawsonia inermis L. & its nano-formulation could act as dual therapy for ulcer treatment and the hazardous effects of aflatoxin exposure.

Keywords: Lawsonia inermis L., Nano-formulation, Ulcer, Aflatoxin, Antioxidant, Milk beverage

1. Introduction

Aflatoxins are one of the most common mycotoxins discovered in the 60tys and categorized by the U.S. Food and Drug Administration (FDA) to be an unavoidable contaminant of foods [1]. Although it's considered the most studied mycotoxin, there's a great lack of information about the risk of aflatoxin exposure to high-risk groups such as people with accompanying diseases.

Peptic ulcers are sores on the stomach, small intestine, or esophageal lining. A stomach peptic ulcer is referred to as a gastric ulcer. A duodenal ulcer is a kind of peptic ulcer that begins during the first part of the small intestine (duodenum). The bottom region of the esophagus is affected by an esophageal ulcer. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) contribute to around 10% of Peptic ulcers disease (PUD) cases. The usage of such medications has risen considerably in recent decades [2].

Since old times, plants and their bioactive extracts have been used in folk medicine and inherited from ancient civilizations to remedy a variety of ailments and diseases. Presently, herbal treatments are regaining their position as a therapy option other than the synthetic medications that are commercially accessible on, especially with the remarkable safety profile and cost-effective regimens which based on their cheaper cost, perceived efficacy, and accessibility, as well as the fact that it has little or no negative consequences. Many researchers have addressed the protective and curative roles of plant extracts against peptic ulcers, as well as the role of some of these plant extracts in decreasing or (regressing) the hazardous effects of aflatoxin exposure [3], one of the plant extracts that have proven effects in ulcer treatment/protection was Lawsonia inermis L. that showed promising anti-ulcer properties [4].

Nanotechnology has the potential to boost conventional medicine by aiding in the discovery, creation & distribution of a variety of assistance techniques to promote health and minimize the consequences of a variety of illnesses which is being done to improve the technologies that can be used or are being adjusted for traditional medical research. By showcasing these technologies, it is anticipated that the potential for nanomaterials to change conventional medical research will be acknowledged [5,6].

Milk beverages are among the most satisfying and nutritious beverages. They're also a great way to transport bioactive components like phenols once isolated from various sources [7]. Milk contains a variety of bioactive peptides, including peptides that help with digestion. Biological studies stated that dairy products offer gastro protective qualities; whey protein in milk has been demonstrated to protect the stomach mucosa in several investigations [8,9]. Bovine whey protein reduced ulcerative lesions by 50.1 and 44%, respectively [10]. α –lactalbumin is one of the most vital proteins in milk that inhibit ulcers by lowering gastric damage, increasing prostaglandin synthesis, and increasing mucin contents of both the gastric fluid and adherent mucus gel layer.

Many researchers have addressed the protective and curative roles of plant extracts against peptic ulcers, as well as the role of some of these plant extracts in decreasing or (regressing) the hazardous effects of aflatoxin exposure [[3], [11]][11, 3], one of the plant extracts that have proven effects in ulcer treatment/protection was Lawsonia inermis L. that showed promising anti-ulcer properties [4,12]. So., in this regard, the current work aimed to study the ameliorating effect of Lawsonia inermis L. & its nano-formulation versus aflatoxin ingestion in ulcerative rats, taking into consideration the fact that aflatoxin exposure to stressed rats (ulcerative) has not been studied before and this is first research addressing such an issue and also, application in milk beverage.

2. Materials & methods

2.1. Materials

Lawsonia inermis L. leaves had been acquired from the Experimental Farm, the Medicinal and Aromatic Plants Research Department, El-Kanater El-Khaireya, Horticulture Research Institute, and Agriculture Research Centre and carefully dried at room temperature moreover grounded to powdery with a mechanical Miller. The powder had been preserved in a glass container at room temperature. Misr for Food Additives (MISAD), Giza, Egypt, provided the emulsifier mono and diglyceride 60%, while pectin was from Sisco Research Laboratories (SRL) in Mumbai, India. Commercial quality granulated cane sugar had been acquired at the local store. It was provided manufactured by Sugar and Integrated Industries. The rest of the chemicals and solvents were obtained from Co. In Hawamdia. MERCK, USA.

2.2. Methods

2.2.1. Extraction of the volatile oil

Extraction of the volatile oil from the Lawsonia inermis L. was done according to [3].

2.2.2. Extraction of Lawsonia inermis L. Phenolics

Lawsonia inermis L. phenolics’ extract was assessed according to [9] method.

2.2.3. Identification of Lawsonia inermis L. Components by chromatography-mass spectrometer (GC/MS)

The qualitative and quantitative analyses of Lawsonia inermis L. were carried out by using the GC system, an Agilent 7890 A with a split injector port set to 200 °C (a split ratio of 200:1) according to [13]. All calibration curves and sample concentrations were created using the (B.09.00) Mass-Hunter software.

2.2.4. Identification of Lawsonia inermis L. Phenolics

Phenolic identification was carried out by HPLC analysis (Agilent 1260 series).

2.2.5. Determination the antioxidant activity

DPPH assay had been measured to investigate antioxidant scavenging activity. IC50 was being used to express antioxidant activity where Lawsonia inermis L. concentration was necessary to elicit a 50% reduction in the initial DPPH concentration [14]. As a reference, vitamin C had been used.

ABTS scavenging capacity of samples was stated by [15]. The scavenging capacity of free radicals was measured by applying vitamin C as a reference. IC50 (mg/L) indicates the dose necessary to scavenge 50% of ABTS radicals.

2.2.6. Antimicrobial assays

2.2.6.1. The tested microorganisms' strains of bacteria and fungi

The Agro-food microbial culture collection provided all the bacterial & fungal strains included in this study, Italy's Institute of Sciences of Food Production (ISPA). Enterococcus faecium, Staphylococcus aureus ATCC 33591, Bacillus subtilis NCIB 3610, Klebsiella pneumoniae NCIB 418, Escherichia coli NCIB 86, and Salmonella senftenberg ATCC 8400 were obtained from TCS Bioscience LTD, Botolph Claydon. Buckingham, MK1821 R. The toxigenic fungi strains included Aspergillus flavus ITEM 698, Aspergillus parasitics ITEM 11, Penicillium chrysogenum ATCC 48271, and Fusarium culmorum KF846.

2.2.6.2. Microorganisms' growth media

For bacteria; nutrient agar (NA) and trypticase soy broth (TSB) were applied to reactivate and enrich the utilized bacterial strains. In contrast, trypticase soy agar (TSA) was applied in the experimental study. For fungi; potato dextrose agar (PDA) and Sabouraud Dextrose agar (SDA) were utilized for fungal strains reactivating. The fungal strains were reactivated using the SDA media with chloramphenicol. It was maintained using PDA media with chloramphenicol. For bacterial strains, they were cultured and activated using Tryptic soy broth media at 37 °C/36 h and were maintained using NA media at 4 °C until the antimicrobial test.

2.2.6.3. Conidial suspension of fungi preparation

On the seventh day of the culture period, tween 80 aqueous solution (0.01%) sterile was used to harvest conidia from culture plates and scrap the culture plates with a bent glass rod to assist conidia's emission from the plates. The number of conidia was adjusted to approximately 106 conidia/mL using a Burker-Turk counting chamber (Heamocytometer).

2.2.6.4. Determination of antifungal and antibacterial activity

Antimicrobial activity was assessed using an agar diffusion assay, as stated by [16]. The incubation condition was at 37 °C for 24 h for bacteria and yeast but was 25 °C for 96 h for fungi. The test susceptibility was obtained by measuring the zone inhibition dimension around the well. The antibiotic gentamicin (30μg/ml) and the antifungal Nystatin (30μg/ml) were used for antibacterial and antifungal activity.

2.2.6.5. The determination of the minimal concentration of inhibitory and antifungal

The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the prepared extract were determined as described by Ref. [17]. As stated by Ref. [18], minimal antifungal concentration (MFC) was evaluated.

2.2.7. Preparation of nano-formulation

Nano-formulation had been prepared using the solvent emulsification-diffusion process of creative modifications [19]. This study has an under-reviewed patent (Number: 1956/2020) by the Egyptian Patent Office, Academy of Scientific Research & Technology, which covers the nano preparation process as well as the relevant measurements data.

2.2.8. Characterization of nano-formulation

Lawsonia inermis L. nano-formulation was assayed for size distribution (by number), polydispersity index (PDI), and ζ-potential via photon correlation spectroscopy at 25 °C; using a Zeta Sizer Nano ZS (Malvern Instruments Inc., Southborough, MA). A TEM (JEM-1234) with a 120 KV operating voltage, 600,000× magnification power, 0.3 nm resolving power, a CCD camera & a programmed heating/cooling facility varying from −190 °C to 1000 °C had been used to investigate the samples which maintained on a carbon-coated copper grid.

2.2.9. Experimental animals

Seventy two male Sprague-Dawley rats between the ages of 1 and 2 months weighing 150–200 gm were kept constant on a basic synthesized diet & water for 7 days prior to the experiment to allow for acclimatization and normal development & behavior. Individual solid bottom cages were used to acclimate the rats in a temperature-controlled (23 °C), 40–60 g/100 g absolute moisture, and light (12 h dark/light cycle) environment free of chemical contamination.

The Egyptian National Research Centre's Ethical Committee of Medical Research approved the animal experiment (approval no. 9997082022) in compliance with the UK's Animals (Scientific Procedures) Act, 1986 and accompanying recommendations, as well as EU Directive 2010/63/EU for animal studies (Publication No. 85–23, revised 1985). The study is reported in accordance with ARRIVE guidelines.

2.2.10. Diet composition

The basic synthesized diet included casein (150 g/kg diet), unsaturated fat (100 g/1 kg diet), sucrose (220 g/kg diet), maize starch (440 g/kg diet), cellulose (40 g/kg diet), salt mixture (40 g/kg diet), and vitamin mixture (10 g/kg diet) [[20], [21]]. The AIN-93 M diet created the salt and vitamin mixtures [22].

2.2.11. Experimental design

Seventy two rats were randomized into the following 12 groups, each with six rats: Negative Control: Normal rats were given the basic synthesized diet. Group (1): Rats were given the basic synthesized diet supported by Lawsonia inermis L. extract at a daily dosage (200 g/kg) [23]. Group (2): Rats were given the basic synthesized diet supported by Lawsonia inermis L. nano-formulation daily dosage (200 g/kg) [23]. Group (3) (positive control): Peptic ulcerated rats were given the basic synthesized diet. Each rat was administered a single dose of dissolved aspirin in water (500 mg/kg body weight) orally [[24], [25]]. Group (4): Peptic ulcerated rats, administered a single dose of dissolved aspirin in water (500 mg/kg body weight) orally, were given the basic synthesized diet supported by Lawsonia inermis L. extract daily dosage (200 g/kg). Group (5): Peptic ulcerated rats, administered a single dose of dissolved aspirin in water (500 mg/kg body weight) orally, were given the basic synthesized diet supported by Lawsonia inermis L. nano-formulation with a daily dosage (200 g/kg). Group (6): Rats, given a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage [26], were given the basic synthesized diet. Group (7): Rats, given a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage, were given the basic synthesized diet supported by Lawsonia inermis L. extract daily dosage (200 g/kg). Group (8): Rats, given a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage, were given the basic synthesized diet supported by Lawsonia inermis L. nano-formulation with a daily dosage (200 g/kg). Group (9): Peptic ulcerated rats receiving a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage were given the basic synthesized diet. Group (10): Peptic ulcerated rats receiving a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage were given the basic synthesized diet supported by Lawsonia inermis L. extract daily dosage (200 g/kg). Group (11): Peptic ulcerated rats receiving a daily dosage (80 μg/kg b. w) of aflatoxin via oral gavage were given the basic synthesized diet supported by Lawsonia inermis L. nano-formulation daily dosage (200 g/kg).

2.2.12. 2.2.12. blood sample collection

Upon completion of the two-month study period, all animals were starved for 12 h. A blood sample was obtained from each animal's tail to test biochemical parameters with a concentration of around 5 ml before being anesthetized with ketamine hydrochloride (35 mg/kg, i. m) then euthanized by cervical dislocation. Serum and plasma had been separated at 4000 rpm for 15 min (Sigma Labor Centrifuge GMBH, West Germany, model 2–153360 osterode/Hertz) and stored at −20 °C.

2.2.13. 2.2.13. gastric juice and pH measurement

The gastric juice was collected and centrifuged for 10 min At 3000 rpm, and the volume was ascertained in mL/100 g BW. A digital pH meter had been applied to evaluate the supernatant's pH [27].

2.2.14. 2.2.14. determination of free acidity and total acidity

Total and free acidity had been calculated in mL of 0.1 N HCl/100 g of gastric contents, corresponding to mEq/L as stated by [28]. The acidity formula was applied:

Acidity = Volume of NaOH × Normality of NaOH × 100/0.1 mEq/lit.

2.2.15. 2.2.15. biochemical parameters

The lipid profiles, including total cholesterol, HDL, LDL, and triglycerides, were ascertained using enzymatic colorimetric methods [[29], [30], [31], [32], [33]]. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total plasma protein, and plasma albumin (A) were ascertained via colorimetric techniques [[34], [35], [36], [37]]. Plasma globulin (G) was calculated. Plasma malondialdehyde (MDA), plasma catalase activity, and total plasma antioxidants were ascertained by colorimetric methods [[38], [39], [40]]. Colorimetric methods were applied to ascertain creatinine, urea, and uric acid [[41], [42], [43]].

2.2.16. 2.2.16. preparation of milk beverage fortified with Lawsonia inermis L. Nanoformulation as application

As stated by [[9], [44]], the milk beverage formulations were prepared. Skimmed milk powder (12% total solids) was reconstituted in distilled water, and Lawsonia inermis L. nanoformulation was added with different ratio to equivalent 100, 200, 300, 400 and 500 mg phenolics/100 ml milk. Each formula concludes with sweetener (sucrose 5% w/v), stabilizer (pectin 0.1% w/v). For 15 s, the milk beverage formulas were pasteurized at 72 °C. All milk beverages were filled in sanitized bottles (100 ml) and kept at 4 ± 1 °C.

2.2.17. 2.2.17. chemical and physical analysis of the milk beverage

The total solid fat, protein, ash, lactose content and titratable acidity of samples have been determined using [20] standard protocol. A pH meter (HANNA Instruments, microprocessor pH 211 UK) was used to assess the pH of samples shortly after Processing at 25 °C.

2.2.18. 2.2.18. determination of total phenolic content (TPC) and radical scavenging activity (RSA) analysis

The total phenolics content was assessed as stated by [45], and the findings were represented as mg of catechin equivalent/ml. To assess DPPH radical-scavenging activity, the method stated by [46] was applied, and the findings were represented as Inhibition%.

2.2.19. 2.2.19. sensory evaluation

The milk beverages were subjected to sensory evaluation, as [47] stated. For sensory evaluation, a sample of 10–15 ml was served. The 10–12 qualified panelists evaluated the Dairy Department, Institute of Food Industry Research and Nutrition, Egypt. Drinking water was used to rinse the mouth and reduce the carryover effect. The sensory attributes of appearance, flavor, body, texture, as well as overall acceptability were assessed using a descriptive score-card with a 10-point intensity scale, with 1 mark for the lowest intensity and 10 marks for the maximum intensity of each sensory attribute. This study was approved by the Egyptian National Research Centre's Ethical Committee of Medical Research (approval no. 9997082022).

2.2.20. 2.2.20. statistical analysis

SPSS/PC software program (version 22.0; SPSS Inc., Chicago, IL, USA) was used to analyze the data (mean values, standard deviation, and standard error). A one-way analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05) was used to determine the significance of differences at 5%.

3. Results and discussion

Mycotoxins, especially aflatoxin, are a universal food safety alarm categorized by the World Health Organization (WHO). Although there is great research legislation, governmental efforts, and control measures, the control of such a problem is still far from reach, especially in agricultural communities in developing countries, being the populations most at risk of aflatoxin exposure. According to the Food and Agriculture Organization (FAO) [48], mycotoxins impact one-fourth of all crops worldwide. Aspergillus, Fusarium, and Penicillium seem to be the three principal genera of fungi that yield mycotoxins [49]. We did not find reports about aflatoxin poisoning in ulcerative cases during our work, so we aimed to shed some light on such cases and the probable protective role of Lawsonia inermis L. extract for both diseases.

3.1. Identification of essential oil from Lawsonia inermis L

Chromatography is frequently used to quantify and qualitatively analyze mixtures and purify chemicals, and determine thermochemical constants such as temperatures of solution and vaporization, vapor pressure, and activity coefficients. Discovering new sources of economic phyto-compounds to synthesize complicated chemical substances and learning the true importance of traditional cures may benefit from understanding plants' chemical contents. This knowledge is important not just for identifying medicinal agents but also for other reasons. Higher plants continue to serve a key role in human health maintenance as sources of bioactive chemicals [50]. Our chemical content of Lawsonia inermis L. is presented in Table 1 an agreement with [51]. GC-MS analysis of Lawsonia inermis L. ethanolic extract showed the presence of nine major compounds as follows: d-allose (18.97%), Lawsone (12.87%), β-d-glucopyranoside, methyl (12.74%), Phytol (10.98%), 1-isobutoxy-1-methoxypropane (9.37%), n-hexadecanoic acid (6.35%), 9,12,15-octadecatrienoic acid (Z, Z, Z) (7.53%), Squalene (8.15%) and vitamin E (7.65%).

Table 1.

Major chemical compounds in essential oil from Lawsonia inermis L.

Compounds	Content (%)
d-allose	18.97
Lawsone	12.87
β-d-glucopyranoside, methyl	12.74
Phytol	10.98
1-isobutoxy-1-methoxypropane	9.37
n-hexadecanoic acid	6.35
9,12,15-octadecatrienoic acid (Z,Z,Z)	7.53
Squalene	8.15
Vitamin E	7.65

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3.2. Identification of Lawsonia inermis L. Phenolics

The chromatogram of the major phenolic compounds in Lawsonia inermis L. phenolic using HPLC, was presented in Table 2. The concentration of Gallic acid, Catechin, Ferulic acid, Chlorogenic acid, Naringenin, Coffeic acid, Rutin, Pyro catechol, Syringic acid were 55823.05, 44757.48, 11864.85, 9662.99, 7951.75, 7013.10, 6347.09, 4816.16, 2410.86 μg compound/g extract followed by other phenolics in minor concentrations. The extract exhibits high antioxidant activity which was more in harmony with [52] despite the differences as in all previous reports of Lawsonia inermis L. extracts.

Table 2.

HPLC analysis of Lawsonia inermis L. phenolics extract.

Phenolics compounds	Concentration (μg/g)
Gallic acid	55823.05
Catechin	44757.48
Ferulic acid	11864.85
Chlorogenic acid	9662.99
Naringenin	7951.75
Coffeic acid	7013.10
Rutin	6347.09
Pyro catechol	4816.16
Syringic acid	2410.86
Kaempferol	402.60
Coumaric acid	352.17
Taxifolin	263.46
Vanillin	161.14
Methyl gallate	114.39
Cinnamic acid	93.19

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3.3. Antioxidant activity

DPPH and ABTS results presented in Table 3 were consistent with [53] which revealed that Lawsonia inermis L. possesses higher antioxidant activity than ascorbic acid, no matter the solvent used. Nevertheless, the ethanol extract showed the highest antioxidant activity, followed by the aqueous extract, and the petroleum ether extract had the least antioxidant activity. The high antioxidant activity in the tested Lawsonia inermis L. extracts could be mainly attributed to the high content of gallic acid (55823.05 μg/g) (Table 2) and lawsone (12.87%) (Table 1) which possess high antioxidant activities. The diverse mechanisms of radical-antioxidant interactions in these tests might explain the various scavenging activities of the extracts against DPPH and ABTS radicals.

Table 3.

Lawsonia inermis L. extracts antioxidant activity.

Type of extract	DPPH assay IC₅₀ (mg/L)	ABTS assay IC₅₀ (mg/L)
Aqueous	19.9 ± 0.71	7.4 ± 0.65
Ethanol	16.4 ± 0.85	6.8 ± 0.53
Petroleum ether	165.5 ± 0.75	744.2 ± 8.62
Vit. C	5.3 ± 0.51	1.9 ± 0.13

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Values are expressed as Mean ± SEM (SEM: standard error mean; n = 3).

3.4. Antimicrobial activity

The phenolics component of plant extract is responsible for most of the antibacterial action. The distinction between Gram + ve and Gram-ve bacteria's sensitivity to polyphenols is a contentious topic. On the one hand, numerous scientists determined that polyphenols' antibacterial activity was typically more efficient against Gram + ve bacteria than Gram-ve bacteria. Because Gram-ve bacteria have a cell wall coupled to a complex outer membrane, they are more resistant to plant secondary metabolites such as phenolics. However, certain polyphenols may affect the outer membrane [54], and the current study's finding adds additional support to such a claim. In this study, we assessed the use of Lawsonia inermis L. and its nano-formulation and antimicrobial properties.

•
The results were expressed in mean ± SEM (SEM: standard error mean; n = 3).
•
The extract's inhibition zone against the bacterial growth was measured in millimeters.
•
G + ve referred to gram-positive bacteria; G-ve referred to gram-negative bacteria.
•
MIC: the minimal inhibition concentration of applied extract against an examined bacterial strain was measured as 1 mg of the extract per milliliter of bacterial growth media.
•
MFC: the minimal fungicidal concentration of an applied extract against an examined fungal strain was measured as a milligram of the extract per milliliter of fungal growth media.

The antibacterial activity results of Lawsonia inermis L. extract in Fig (1: A, B, C, D) revealed that this extract could be effective against various microorganisms as G+ pathogenic bacteria were more affected by Lawsonia Inermis L. extract than G-ve, with Staph aureus being the most affected strain, followed by Enterococcus faecium and B. subtilis, respectively, where the MIC was 420, 450, and 450 ng/ml. The same pattern was recorded for MBC, 530, 550, and 570 ng/ml. While for G-ve, all the tested strains were affected by the extract Salmonella senftenberg and Klebsiella pneumoniae, where the MIC was 500 ng/ml, and for Escherichia coli was 520 ng/ml which support previous reports on Lawsonia inermis L. antimicrobial properties, which were in good harmony with [4]. The antimicrobial activity of Lawsonia inermis L. nanomulsion in Fig (1: E, F, G, H) was evaluated against pathogenic bacteria, either G + ve (Fig. 1: A & B) or G-ve (Fig. 1: C & D) and the results revealed that G + ve pathogenic bacteria were more affected by Lawsonia Inermis L. nano-formulation than G-ve, as was the case for the normal extract. Nevertheless, the nano-formulation exhibited higher antibacterial activity, with B. subtilis being the most affected strain, followed by Enterococcus faecium and Staph aureus, respectively. The MIC was 280, 310, and 330 ng/ml, and MBC recorded 320, 300, and 330 ng/ml. While for G–ve, all the tested strains were affected by the nano-extract with the same trend as the normal extract for Salmonella senftenberg and Klebsiella pneumoniae, where the MIC was 360 ng/ml, and for Escherichia coli was 350 ng/ml which has shown higher susceptibility to the tested strains, providing safety and commercial advantage for using the nano-formulation. This advantage could be due to the nanoscale particle size, which facilitates the movement of the plant extract particles within the microorganism that can cause much more harm to the microorganism than the normal form. The antifungal properties of Lawsonia inermis L. extract were examined contra four toxigenic fungal strains: Aspergillus flavus, Aspergillus parasiticus, Fusarium culmorum, and Penicillium verrucosum. The obtained results are illustrated in Fig. (1: I & J), where Fusarium culmorum was the most affected strain even in a low concentration as low as 50 μg, followed by Penicillium verrucosum, then Aspergillus flavus, and Aspergillus parasiticus, and the recorded MFCS were 0.27, 0.3, 0.39, and 0.55 mg/ml, respectively. The high antioxidant activity in the tested Lawsonia inermis L. extracts could be mainly attributed to the high content of catchin (44757.48 μg/g) (Table 2) and lawsone (12.87%) (Table 1) which possess high antimicrobial activities. The antifungal properties of Lawsonia inermis L. nano-formulation were examined contra four toxigenic fungal strains: Aspergillus flavus, Aspergillus parasiticus, Fusarium culmorum, and Penicillium verrucosum. The results are illustrated in Fig. (1: k & l), where Fusarium culmorum was the most affected strain. The antimicrobial activity of the nano-formulation did not differ markedly from the normal extract. The results of the antifungal activity of Lawsonia inermis L. and its nano-formulation in Fig (1: I, J, K, L) support the previous hypothesis as there were higher antifungal activities of Lawsonia inermis L. nano-formulation in all the tested toxigenic fungal strains, and the antifungal response was dose-dependent in all the tested strains, and these findings corroborated prior works [55].

3.5. Lawsonia inermis L. Nano-formulation characterizations

Nanomaterials have a greater surface area than their bulk counterparts with nanoscale effects, making them viable plant improvement tools. Owing to their peculiar size, shape, chemical composition, surface structure, charge, solubility, and agglomeration, nanomaterials differ from their bigger counterparts in terms of physicochemical and biological characteristics, which may have a big impact on how they interact with biomolecules and cells. With nano-medicine, early detection and prevention, better diagnosis, correct treatment, and follow-up of illnesses are achievable. The nanoparticles' large surface area to volume ratio is unique when the nanoparticle size ranges from 10 to 100 nm.

TEM, particle size and ζ-potential (Fig. 2 A, B & 3) were demonstrated for investigating the properties and morphology of Lawsonia inermis L. nano-formulation, which confirmed the existence of Lawsonia inermis L. nano-formulation with diverse particle diameters below 100 nm. The particle sizes were estimated to be about 7–20 nm with round and spherical shape. The Zeta Sizer Nanoparticle characterization system measures particle size, ζ-potential, and molecular weight. Depending on Zeta Sizer result for Lawsonia inermis L. nano-formulation which were Z-average (d.nm) = 50.3, Pdl = 0.24, particle size (d.nm) = 22.7 with % Number = 99.9% and ζ-potential = −83.7, we confirmed that Lawsonia inermis L. are nanoparticles with a nanoscale of less than 100 nm and a very uniform distribution observed (Fig. 3). The magnitude of the ζ-potential of a colloidal system is linked to its physical stability; if particles in a solution have a large ζ-potential (negative or positive); they repulse each other and reduce the probability of forming aggregations. Particles with ζ-potential greater than 30 mV (positive or negative) are regarded as physically constant. In contrast, ζ-potential close to 20 mV (positive or negative) indicates low colloidal suspension steady, and rapid particle aggregation may be included in the range of −5 mV to +5 mV [[56], [57]].

Fig. 3 — The particle size, PDI and ζ-potential of *Lawsonia inermis* L. nano-formulation.

3.6. The biological study

The biological study in the present work is multifactorial, and it involves three main branches. The 1st is the effect of aflatoxin exposure on ulcerative rats; the 2nd is the protective effect of Lawsonia inermis L. extract and its nano-formulation as remedies for ulcerative rats, and the 3rd is the ameliorative effect of Lawsonia inermis L. extract and its nano-formulation for ulcerative rats exposed to aflatoxin. The anti-ulcerative Activity of Lawsonia inermis L. extract results presented in Table 4 indicated a significant elevation in all the gastric juice, pH, free acidity, and total acidity in the aflatoxin-treated group [[4], [58]]. These results could regard aflatoxin as a possible causative factor for ulcer development, yet more research is needed to clarify this hypothesis. The results also showed higher records of all the tested parameters in the ulcerative aflatoxin treated group, which could give clear evidence that exposure to aflatoxin deteriorates the ulcerative case in the ulcer suffering rats. No previous reports discussed the role of aflatoxin in ulcer induction or combination with aflatoxin exposure in ulcerative rats. Using Lawsonia inermis L. in, both normal and nano-formulation caused significant enhancements in each evaluated parameter, including gastric juice, pH, free acidity, and total acidity toward the normal values, which was evident in the beneficial effect of Lawsonia inermis L. extract in managing the ulcerative parameters. These results were much more evident in the ulcerative groups. There was significant advancement throughout the tested parameters toward normal. These results support previous results as [4].

Table 4.

Effect of Lawsonia inermis L. extract and its nano-formulation on volume of Gastric juice, pH, Free Acidity and Total Acidity.

Groups	Volume of Gastric juice (mL)	pH	Free Acidity meq/L/100 g	Total Acidity meq/L/100 g
Negative Control	2.35 ± 0.18	2.33 ± 0.19	26.32 ± 0.87	24.67 ± 2.55
Group (1)	2.22 ± 0.56^a	2.44 ± 0.15^a	24.48 ± 0.52^a	23.46 ± 2.62^a
Group (2)	2.15 ± 0.48^a	2.56 ± 0.03^a	23.33 ± 0.59^a	22.57 ± 2.46^a
Group (3)	3.7 ± 0.18^b	1.34 ± 0.39^b	59.8 ± 0.43^b	81.12 ± 2.78^b
Group (4)	1.91 ± 0.11^c	2.27 ± 0.18^c	24.36 ± 0.61^c	42.11 ± 2.39^c
Group (5)	1.51 ± 0.13^d	2.19 ± 0.19^d	24.88 ± 0.53^d	40.31 ± 2.22^d
Group (6)	3.13 ± 0.14^e	2.13 ± 0.23^e	54.84 ± 0.37^e	74.79 ± 2.86^e
Group (7)	1.74 ± 0.15^f	2.22 ± 0.16^f	24.56 ± 0.48^f	43.15 ± 2.56^f
Group (8)	1.99 ± 0.14^g	2.23 ± 0.17^g	25.16 ± 0.24^g	42.32 ± 2.23^g
Group (9)	3.93 ± 0.16^h	2.06 ± 0.37^h	61.32 ± 0.56^h	79.68 ± 2.19^h
Group (10)	1.43 ± 0.12ⁱ	2.11 ± 0.12ⁱ	31.45 ± 0.26ⁱ	41.69 ± 2.22ⁱ
Group (11)	1.59 ± 0.12^j	2.13 ± 0.14^j	28.12 ± 0.13^j	39.34 ± 2.36^j

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Values are expressed as Mean ± SEM (n = 6) in which the same letters in each column imply a non-significant difference across varieties, whereas different letters imply a significant difference at P ≤ 0.05.

The investigated nutritional parameters in Table 5 revealed no significant difference in all groups' initial body weight or final body weight. There is also no significant notice in body gain, total food intake, and feed efficiency in groups (1, 2) in contrast to normal control. There're non-significant elevations in body gain and feed efficiency where there's a significant decrease in total food intake in group (3) in contrast to normal control. There's a significant decrease in body gain and feed efficiency, whereas there's a significant increase in total food intake in groups (4, 5) in comparison to group (3). There's a significant decrease in body weight gain, feed efficiency, and total food intake in group (6) in contrast to normal control. There's a significant decrease in body gain and feed efficiency and a significant increase in total food intake in groups (7, 8), in contrast to the group (6). No significant differences appeared in body gain, feed efficiency, and total food intake in the group (9) in contrast to the group (6). There's a significant decrease in body weight gain and feed efficiency where there's a significant increase in total food intake in groups (10, 11) in contrast to group (9). The decrease in total food intake in the ulcer treated groups that may have contributed to the deterioration in the physiological stomach status of the animals, which was also reflected in body weight gain; these parameters were improved in the Lawsonia inermis L. extract treated groups [4].

Table 5.

Effect of Lawsonia inermis L. Leaves in normal and nano scale on initial body weight, final body weight, body gain, total food intake and feed efficiency.

Group	Initial body weight (g)	Final body weight (g)	Body gain (g)	Total food intake (g)	Feed efficiency
Negative Control	195.2 ± 1.78	245.8 ± 3.85	50.7 ± 2.28	1192.2 ± 1.64	0.042 ± 0.002
Group (1)	192.8 ± 1.66^a	245.5 ± 3.28^a	52.7 ± 1.94^a	1192 ± 2.56^a	0.044 ± 0.002^a
Group (2)	191.7 ± 2.17^a	246 ± 4.86^a	54.3 ± 3.22^a	1194.7 ± 1.58^a	0.045 ± 0.003^a
Group (3)	192.3 ± 2.65^a	237.5 ± 4.29^a	45.2 ± 2.52^a	1147.2 ± 7.33^b	0.039 ± 0.002^a
Group (4)	191 ± 2.48^a	229 ± 3.64^a	38 ± 1.39^b	1184.7 ± 2.11^c	0.032 ± 0.001^b
Group (5)	190.2 ± 1.85^a	227.8 ± 4.53^a	37.7 ± 2.73^c	1185.2 ± 1.78^d	0.032 ± 0.002^c
Group (6)	192.8 ± 2.18^a	237.7 ± 4.22^a	44.8 ± 2.29^d	1148 ± 7.66^e	0.039 ± 0.002^a
Group (7)	191 ± 2.05^a	230.7 ± 2.84^a	39.7 ± 1.38^e	1185.3 ± 1.52^f	0.033 ± 0.001^d
Group (8)	190.2 ± 2.41^a	228.8 ± 4.43^a	38.7 ± 2.23^f	1185.2 ± 1.78^g	0.033 ± 0.002^e
Group (9)	192.2 ± 2.21^a	239.2 ± 3.76^a	47 ± 2.41^a	1148.8 ± 7.85^a	0.041 ± 0.002^a
Group (10)	190.7 ± 2.42^a	230.7 ± 2.84^a	40 ± 0.93^g	1185.5 ± 1.38^h	0.034 ± 0.001^f
Group (11)	190.2 ± 1.76^a	229.5 ± 5.43^a	39.3 ± 3.88^h	1185.2 ± 1.78ⁱ	0.033 ± 0.003^g

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Assessment of the biochemical parameters in the tested groups revealed that the lipid profile in Table 6 showed significant elevation in the ulcer treated group and aflatoxin treated group compared to the normal; that pointed out by Ref. [59]. They explained the association of high lipid profile in ulcerative patients, especially those that H pylori-infected, which could be the case during ulcer induction, while the disturbance in lipid profile in aflatoxin treated rats supports prior study [60] that is due to the harmful effects of aflatoxin that target the liver and disrupt its performance. Most of the harmful action was due to oxidative stress regarding ulcer or aflatoxin exposure. This hazardous effect was rolled out by the antioxidant activity presented by Lawsonia inermis L. extracts. That was apparent in the remarkable, significant improvement in the lipid profile in all the treated groups, either with Lawsonia inermis L. or nano-formulation. These findings corroborate prior work [4]. By investigating the effects on rat liver, results in Table 7 indicated a significant increase in liver enzymes in the ulcer group alone and the aflatoxin and aflatoxin + ulcer treated groups, which was evident in the high toxic load on the liver, leading to liver stress and causing liver cell degeneration that was reflected in the elevation of all the tested liver parameters. This harmful sequence is due to free radical generation that strongly attacks the liver, leading to liver damage [61]. Several studies have shown that antioxidants may reduce the hepatotoxicity risk induced by aflatoxin exposure [56]. The obtained results support the same claim, so the improvement in all the measured parameters in the groups given Lawsonia inermis L. or nano-formulation is due to the antioxidant capacity that Lawsonia inermis L. extract possesses. Our study results in Table 7 matched with [62] which showed elevated urea and creatinine blood levels of Nile tilapia subjected to different concentrations of AFB1. The serum total protein and albumin levels in Table 6 decreased in the same observation found in the present study. There were no signs in either the ulcer group or the aflatoxin group, which is a major indication of serious liver deterioration.

Table 6.

Effect of Lawsonia inermis L. extract and its nano-formulation on Total cholesterol, HDL, LDL,Triglycerides, Catalase, Lipid Peroxide, Total Antioxidant Capacity, Albumin, Total Protein and Globulin.

Group	Total cholesterol mg/dL	HDL mg/dL	LDL mg/dL	Triglycerides mg/dL	Catalase mg/dL	Lipid Peroxide mg/dL	Total Antioxidant mM/L	Albumin g/dL	Total Protein mmol/L	Globulin g/dL
Negative Control	36.8 ± 2.04	18.6 ± 1.18	11.9 ± 0.87	68.6 ± 5.51	50.43 ± 1.48	20 ± 1.38	1.08 ± 0.08	2.79 ± 0.02	5.28 ± 0.29	2.49 ± 0.27
Group (1)	34.4 ± 2.35^a	14 ± 1.72^a	14.3 ± 1.94^a	66.9 ± 3.17^a	56.33 ± 1.39^a	16 ± 1.26^a	1.4 ± 0.11^a	2.81 ± 0.07^a	5.09 ± 0.15^a	2.28 ± 0.08^a
Group (2)	31.5 ± 2.21^a	11.6 ± 1.04^b	11.9 ± 2.06^a	52.4 ± 5.39^b	56.45 ± 2.42^b	17 ± 1.18^b	1.6 ± 0.11^b	2.78 ± 0.1^a	4.72 ± 0.26^a	1.94 ± 0.16^a
Group (3)	80.5 ± 3.45^b	44 ± 3.26^c	27 ± 2.56^b	174.9 ± 4.95^c	70.57 ± 0.8^c	28 ± 1.35^c	3.03 ± 0.07^c	2.33 ± 0.47^a	5.67 ± 0.95^a	3.34 ± 0.48^b
Group (4)	31.5 ± 5.82^c	15.9 ± 1.08^d	16.6 ± 3.93^c	70 ± 6.68^d	34.62 ± 1.61^d	19 ± 1.13^d	1.74 ± 0.1^d	2.58 ± 0.03^a	4.78 ± 0.35^a	2.2 ± 0.32^c
Group (5)	25.3 ± 3.35^d	15.8 ± 0.9^e	11.5 ± 1.50^d	66.1 ± 3.87^e	30.72 ± 2.7^e	19 ± 1.21^e	1.17 ± 0.08^e	2.43 ± 0.04^a	4.58 ± 0.43^a	2.15 ± 0.39^d
Group (6)	82.8 ± 4.24^e	44.9 ± 3.32^f	28.7 ± 3.22^e	180.8 ± 1.23^f	72.37 ± 0.96^f	29 ± 1.28^f	3.03 ± 0.07^f	2.36 ± 0.49^a	5.88 ± 0.5^a	3.52 ± 0.01^e
Group (7)	33.8 ± 5.29^f	15.9 ± 1.34^g	18.2 ± 3.08^f	70.6 ± 6.29^a	34.62 ± 1.61^g	20 ± 1.24^g	1.62 ± 0.09^g	2.62 ± 0.03^a	4.68 ± 0.36^a	2.06 ± 0.33^f
Group (8)	27.6 ± 3.32^g	15.8 ± 1.45^h	11.5 ± 1.5^j	66.1 ± 3.87^g	30.72 ± 3.4^h	18 ± 1.48^h	1.16 ± 0.09^h	2.47 ± 0.06^a	4.63 ± 0.32^a	2.16 ± 0.26^g
Group (9)	87.4 ± 3.35^a	47.8 ± 2.23^a	30.3 ± 2.71^a	184.4 ± 6.29^h	72.90 ± 0.78^a	30 ± 1.15^a	3.12 ± 0.07^a	3.08 ± 0.35^a	6.71 ± 0.42^a	3.63 ± 0.07^a
Group (10)	36.1 ± 4.47^h	15.9 ± 1.57ⁱ	16.6 ± 3.71^h	71.2 ± 5.93^a	34.27 ± 1.62ⁱ	19 ± 0.61ⁱ	1.57 ± 0.09ⁱ	2.62 ± 0.03^a	5.72 ± 0.51^a	3.1 ± 0.48^h
Group (11)	29.9 ± 2.48ⁱ	15.8 ± 0.9^j	13.2 ± 1.44ⁱ	66.7 ± 3.44ⁱ	29.28 ± 1.89^j	18 ± 1.35^j	1.08 ± 0.08^j	2.47 ± 0.06^a	5.44 ± 0.67^a	2.97 ± 0.61ⁱ

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Table 7.

Effect of Lawsonia inermis L. Leaves in normal and nano-scale on Creatinine, Urea, Uric Acid, AST, ALT, and ALP.

Group	Creatinine mg/dL	Urea mg/dL	Uric Acid mg/dL	AST mg/dL	ALT μg/dL	ALP IU/L
Negative Control	0.58 ± 0.05	40.1 ± 4.38	2.45 ± 0.13	33 ± 1.51	26 ± 2.31	65.44 ± 4.92
Group (1)	0.57 ± 0.07^a	39.3 ± 3.97^a	2.37 ± 0.11^a	30.7 ± 1.86^a	23 ± 2.48^a	64.19 ± 4.62^a
Group (2)	0.47 ± 0.02^a	39.2 ± 1.37^a	2.3 ± 0.25^a	27.8 ± 0.98^a	21 ± 2.63^a	63.52 ± 3.9^a
Group (3)	0.96 ± 0.18^b	81 ± 2.37^b	3.7 ± 0.44^b	58.3 ± 0.76^b	38 ± 2.84^b	118.26 ± 9.11^b
Group (4)	0.58 ± 0.05^c	38.9 ± 1.94^c	2.7 ± 0.14^c	37.8 ± 0.95^c	22.3 ± 1.69^c	55.07 ± 2.32^c
Group (5)	0.55 ± 0.05^d	38.1 ± 3.00^d	2.28 ± 0.17^d	35 ± 2.00^d	22 ± 5.16^d	47.48 ± 5.19^d
Group (6)	0.96 ± 0.06^e	79.6 ± 2.08^e	3.85 ± 0.35^e	59.3 ± 0.76^e	37 ± 3.2^e	118.63 ± 8.9^e
Group (7)	0.55 ± 0.06^f	38.5 ± 2.02^f	2.77 ± 0.13^f	38.2 ± 0.87^f	23 ± 3.18^f	56.85 ± 0.89^f
Group (8)	0.54 ± 0.06^g	38.1 ± 2.92^g	2.36 ± 0.19^g	35.3 ± 1.89^g	21 ± 4.78^g	51.19 ± 3.85^g
Group (9)	0.97 ± 0.06^a	83.8 ± 2.23^a	3.93 ± 0.32^a	60.8 ± 2.02^a	40.2 ± 1.22^a	119 ± 5.89^a
Group (10)	0.56 ± 0.06^h	39.2 ± 2.02^h	2.85 ± 0.08^h	38.7 ± 0.99^h	22.5 ± 4.79^h	59.07 ± 2.09^h
Group (11)	0.55 ± 0.06ⁱ	38.8 ± 2.29ⁱ	2.44 ± 0.23ⁱ	35.8 ± 2.17ⁱ	22 ± 5.16ⁱ	50.19 ± 4.1ⁱ

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All these parameters were reversed again via Lawsonia inermis L. extract and its nano-formulation, which is a promising sign of improvement. Results in Table 6 revealed a significant increase in lipid peroxide, catalase, and total antioxidants in the ulcer, aflatoxin, and aflatoxin groups. It is a well-known fact that aflatoxin exhibits its toxic action mainly via the production of free radicals, leading to oxidative stress that was evident in the elevation of lipid peroxidation results [63]. A body's natural defense system responds because an elevation of catalase accompanies that elevation to counteract the harmful effects of the stress conditions the animals are subjected to in the case of ulcers and aflatoxin. Using Lawsonia inermis L. extract and its nano-formulation in the following groups showed that all these records were revised towards the normal measures, meaning the decrease of the oxidative stress conditions via the abundance of antioxidants that even decreased the need for the natural defense (catalase) activity to be higher than normal.

3.7. Application in milk beverage

The chemical, physical analysis, antioxidant activity and total phenolic content of milk beverages fortified with Lawsonia inermis L. nano-formulation were presented in Table 8. The results revealed that there weren't high significant differences between functional beverages that contained different concentrations of Lawsonia inermis L. nano-formulation in total solids, protein, fat, lactose and ash. Total solids were in the range of 12.22–13.30%, the protein was in the range of 4.35–4.80%, fat was in the range of 0.45–0.65%, lactose was in the range of 4.55–4.19%, ash was in the range of 0.84–0.96%, pH was in the range of 6.66–6.30 and the titratable acidity was in the range of 0.18–0.23% for control, T1, T2, T3, T4, and T5, respectively which are compatible with our previous study [9]. Lawsonia inermis L. nano-formulation might have contributed to the increase in chemical composition (p < 0.05) of the milk beverages.

Table 8.

The chemical and physical analysis, total phenolics and the radical scavenging activity of milk beverage fortified with Lawsonia inermis L. nano-formulation.

Samples	Total solids (g/100 ml)	Protein (g/100 ml)	Fat (g/100 ml)	Lactose (g/100 ml)	Ash (g/100 ml)	pH	Acidity (% lactic acid)	Total phenolics (catechin eq. mg/ml)	Radical scavenging activity %
Control	12.22 ± 0.15^d	4.35 ± 0.21^b	0.45 ± 0.07^b	4.55 ± 0.07^a	0.84 ± 0.02^c	6.66 ± 0.05^a	0.18 ± 0.18^b	0.01 ± 0.01^e	3.94 ± 0.03^d
T₁	12.23 ± 0.11^d	4.55 ± 0.07^b	0.40 ± 0.14^ab	4.43 ± 0.04^b	0.91 ± 0.02^b	6.51 ± 0.014^b	0.18 ± 0.18^b	0.48 ± 0.01^d	67.62 ± 0.65^c
T₂	12.40 ± 0.14^cd	4.75 ± 0.07^a	0.55 ± 0.07^ab	4.38 ± 0.03^b	0.93 ± 0.02^ab	6.43 ± 0.014^c	0.19 ± 0.19^ab	0.56 ± 0.03^c	78.48 ± 0.69^b
T₃	12.75 ± 0.21^bc	4.70 ± 0.14^a	0.65 ± 0.07^a	4.40 ± 0.01^b	0.94 ± 0.01^ab	6.39 ± 0.021^cd	0.20 ± 0.20^ab	0.61 ± 0.01^b	79.83 ± 0.90^b
T₄	12.89 ± 0.12^b	4.85 ± 0.07^a	0.65 ± 0.07^a	4.25 ± 0.07^c	0.96 ± 0.01^a	6.35 ± 0.021^de	0.21 ± 0.21^ab	0.64 ± 0.01^ab	82.53 ± 0.13^a
T₅	13.30 ± 0.14^a	4.80 ± 0.14^a	0.65 ± 0.07^a	4.19 ± 0.05^c	0.96 ± 0.01^a	6.30 ± 0.007^e	0.23 ± 0.23^a	0.65 ± 0.01^a	81.41 ± 0.69^a

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All the values are Mean ± SD. ^abMean values in a column with at least one similar superscript do not differ significantly (P < 0.05). Control: milk beverage without Lawsonia inermis L. nano-formulation, T1: milk beverage contains 100 mg phenolics, T2: milk beverage contains 200 mg phenolics, T3: milk beverage contains 300 mg phenolics, T4: milk beverage contains 400 mg phenolics, T5: milk beverage contains 500 mg phenolics.

In the DPPH assay in (Table 8), fortified milk beverage had considerably stronger radical scavenging activity than plain milk beverage (P < 0.05), due to the antioxidants constitutes in Lawsonia inermis L. nano-formulation such as coumarins, flavonoids, gallic acid, naphthalene derivatives and lupane-type triterpenoids. The results show that plain milk contains antioxidant activity, which is mostly due to milk proteins such as caseins, β-LG, α-LA, immunoglobulins, lactoferrin, and serum albumin, which can exhibit antioxidant activity [64]. As stated by [65,66], various milk components, such as cysteine, glutathione, and protein sulfhydryls, can scavenge DPPH radicals based on the amino acids sequence, in agreement with [67]. As presented in Table 8, the addition of Lawsonia inermis L. nano-formulationhas a significant (P < 0.05) increase in the total phenolics values in the milk beverages compared to the plain milk beverage (control). The amount of phenolics in milk beverages with Lawsonia inermis L. nano-formulationranged from 0.48 ± to 0.65 mg of catechin/ml compared to control sample (plain milk beverage), it was 0.01 mg of catechin/ml. due to the high level of phenolic compounds in Lawsonia inermis L. as stated by [14].

Sensory evaluation of the organoleptic properties is significant features such as desirable appearance, flavor, and texture which are of useful preservative characters presented in Table 9. From the results, the milk beverages fortified with different concentrations of Lawsonia inermis L. nano-formulation tends to turn green color as the concentration of the LPE increases. The dark green color obtained suggested that Lawsonia inermis L. leaf biomass predominantly contained dark green coloring pigment. Though variance between the flavors of different milk beverages concentrations, there was no literature that mentioned the flavor features of Lawsonia inermisL. leaf extract, however a comparable intense burning scent was described in America for Western juniper (Juniperus occidentalis) leaf extract [68]. This finding, as well as that of [68], revealed that leaf extracts have a distinct flavor. The result suggested that with increasing the concentration in milk beverage the texture was better due to the leaf's abundant supply of organic soluble oils. Overall acceptability which reflected the general quality of the product indicated that milk beverage with the different ratios of Lawsonia inermis L. nano-formulation was accepted and could be recommended that milk beverage could be fortified with Lawsonia inermis L. nano-formulation up to 500 mg phenolics to get acceptable and high potential health product.

Table 9.

The sensory evaluation of milk beverage fortified with Lawsonia inermis L. nano-formulation.

Samples	Appearance	Flavor	Body and texture	Overall acceptability
Control	9.80 ± 0.34^a	9.81 ± 0.22^a	9.89 ± 0.19^a	29.51 ± 0.59^a
T1	9.70 ± 0.22^a	8.87 ± 0.48^b	8.55 ± 1.07^d	28.00 ± 0.95^b
T2	9.50 ± 0.25^a	7.30 ± 0.75^c	8.75 ± 0.72^cd	26.1 ± 0.90^c
T3	8.70 ± 0.39^b	7.27 ± 0.67^c	8.90 ± 0.52^bcd	24.61 ± 1.20^d
T4	7.80 ± 0.64^c	7.10 ± 0.84^c	9.26 ± 0.42^bc	23.32 ± 1.23^e
T5	7.20 ± 0.70^c	6.80 ± 0.50^d	9.44 ± 0.47^ab	21.30 ± 1.20^f

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All the values are Mean ± SD. abMean values in a column with at least one similar superscript do not differ significantly (P < 0.05). Control: milk beverage without Lawsonia inermis L. nano-formulation, T1: milk beverage contains 100 mg phenolics, T2: milk beverage contains 200 mg phenolics, T3: milk beverage contains 300 mg phenolics, T4: milk beverage contains 400 mg phenolics, T5: milk beverage contains 500 mg phenolics.

4. Conclusion

From the obtained data, this is the first time to document the correlation between aflatoxin exposure and ulcer susceptibility and deterioration that aflatoxin exposure did increase the deterioration in ulcerative rats and vice versa, the ulcerative induction presented a load that worsened the toxic responses to aflatoxin exposure which should be investigated in much more details in the future. The results support the possibility of using Lawsonia inermis L. as a source for the production of possible therapeutic formulation that could act as dual therapy for ulcer treatment and relieve the hazardous effects of aflatoxin exposure.

Author contribution statement

Dina Mostafa Mohammed: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. </p>

Marwa M. El-Said, Bassem Sabry: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper. </p>

Doha H Abou Baker, Ahmed Noah Badr, Amal S Hathout: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. </p>

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Data will be made available on request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data will be made available on request.

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PERMALINK

Promising role of Lawsonia inermis L. leaves extract and its nano-formulation as double treatment against aflatoxin toxicity in ulcerated-rats: Application in milk beverage

Dina Mostafa Mohammed

Marwa M El-Said

Ahmed Noah Badr

Doha H Abou Baker

Amal S Hathout

Bassem A Sabry

Abstract

1. Introduction

2. Materials & methods

2.1. Materials

2.2. Methods

2.2.1. Extraction of the volatile oil

2.2.2. Extraction of Lawsonia inermis L. Phenolics

2.2.3. Identification of Lawsonia inermis L. Components by chromatography-mass spectrometer (GC/MS)

2.2.4. Identification of Lawsonia inermis L. Phenolics

2.2.5. Determination the antioxidant activity

2.2.6. Antimicrobial assays

2.2.6.1. The tested microorganisms' strains of bacteria and fungi

2.2.6.2. Microorganisms' growth media

2.2.6.3. Conidial suspension of fungi preparation

2.2.6.4. Determination of antifungal and antibacterial activity

2.2.6.5. The determination of the minimal concentration of inhibitory and antifungal

2.2.7. Preparation of nano-formulation

2.2.8. Characterization of nano-formulation

2.2.9. Experimental animals

2.2.10. Diet composition

2.2.11. Experimental design

2.2.12. 2.2.12. blood sample collection

2.2.13. 2.2.13. gastric juice and pH measurement

2.2.14. 2.2.14. determination of free acidity and total acidity

2.2.15. 2.2.15. biochemical parameters

2.2.16. 2.2.16. preparation of milk beverage fortified with Lawsonia inermis L. Nanoformulation as application

2.2.17. 2.2.17. chemical and physical analysis of the milk beverage

2.2.18. 2.2.18. determination of total phenolic content (TPC) and radical scavenging activity (RSA) analysis

2.2.19. 2.2.19. sensory evaluation

2.2.20. 2.2.20. statistical analysis

3. Results and discussion

3.1. Identification of essential oil from Lawsonia inermis L

Table 1.

3.2. Identification of Lawsonia inermis L. Phenolics

Table 2.

3.3. Antioxidant activity

Table 3.

3.4. Antimicrobial activity

Fig. 1.

3.5. Lawsonia inermis L. Nano-formulation characterizations

Fig. 2.

Fig. 3.

3.6. The biological study

Table 4.

Table 5.

Table 6.

Table 7.

3.7. Application in milk beverage

Table 8.

Table 9.

4. Conclusion

Author contribution statement

Funding

Data availability

Declaration of competing interest

References

Associated Data

Data Availability Statement

ACTIONS

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RESOURCES

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