Manilkara zapota (M. zapota) antibacterial and antioxidant properties may help speed up the healing process. The present investigation aimed to evaluate the wound healing activity of M. zapota bark ethanolic extract (MZE) by employing in-vitro migration scratch assay and in-vivo animal models. Wistar albino rats were used for the in-vivo wound healing models. No treatment was given to Group I; Group II received povidone-iodine (5% W/W); Group III received MZE (5% W/W); and Group IV received MZE (10% W/W). Linear incision models and excision wound models were used to induce injury. The ointments were applied immediately to the wounds after causing the injury. The percentage of wound contraction, the length of the epithelization period, and the wound’s tensile strength were all calculated. The scratch assay assessed the test drug’s potential for wound healing in-vitro. H2O2 and DPPH scavenging assays were used to measure antioxidant activity. A p < 0.05 was used to define statistical significance. On days 4, 8, 12, 16, and 20, the wound contraction potential of animals treated with MZE ointment was significantly higher (p < 0.001) than that of the control group. On day 20, the proportion of wound contraction in MZE-treated animals was 99.88%, compared to 83.86% in untreated animals. The test group had a significantly (p < 0.01) faster time to full epithelization than the control group. In the incision model, the control group had considerably lower mechanical strength (p < 0.001) than animals treated with MZE. In addition, MZE caused a significant increase (p < 0.001) in total protein and hydroxyproline levels. In the scratch experiment, test drug-treated cells showed a higher rate of cell migration than untreated cells. Furthermore, animals treated with MZE showed increased levels of epithelial tissue, collagen proliferation, and keratinization. To summarize, the current study found that M. zapota improved wound healing activity both in vitro and in vivo, as evidenced by the study results. M. zapota extract has significant wound-healing potential and could be a viable source of wound-healing nutraceuticals.Poor circulation, unresolved inflammation, neuropathy, and infection make wound care difficult.  A wound is defined as damage to the normal structure and function of the skin, resulting from various causes such as trauma, surgery, or underlying medical conditions. Wounds can be classified as either open or closed. This can range from a minor injury to the epithelial tissues of the skin to possibly more severe damage to the subcutaneous tissue and possibly other tissues as well, such as blood vessels, muscles, tendons, bones, parenchymal organs, and nerves . One of Healing of wounds is a spontaneously occurring dynamic process that involves interaction between cytokines, blood, extracellular matrix, and growth factors. The primary objectives of wound management are to stop the bleeding and protect the patient from infection. To effectively treat wounds, it is necessary to use active ingredients that are antioxidant, antibacterial, anti-inflammatory, and promote cell proliferation . CarotenManilkara zapota L., is an evergreen tree in the family Sapotaceae that produces milky juice. The ripe fruits of this plant are edible and have a flavour similar to sugar. M. zapota has been shown to have several medicinal properties, including those that are anti-inflammatory, antipyretic, antitumor, antioxidant, antimicrobial, anti-ageing, anti-lipidemic, and acaricidal  was collected from the Botanical garden of Karnataka University, Dharwad and authentication of the plant was done by qualified Pharmacognosist, Dr. R. A. Shastri, Associate Professor, SETs College of Pharmacy, S. R. Nagar, Dharwad, and a herbarium specimen (SETCPD/HERB/80/I/2022) was preserved. After being dried at room temperature in the shade, it was ground with an electric grinder to obtain a coarse powder.The bark of Manilkara zapota Linn bark of 650 g was used for extraction with 90% ethanol. A solvent ratio of 1:10 was used for the Soxhlet extraction. In every cycle, an estimated quantity of 100 g of dried M. zapota bark powder was used. The heating mantle was set to a temperature of approximately 80°C. The plant material was subjected to 12–24 h of extraction .The Folin-Ciocalteu method  was usedUsing quercetin as a reference standard, the flavonoid concentration in the plant extracts was assessed as per the technique described by FTIR analysis of the dried plant extract was conducted as previously described . To creaM. zapota ethanol extract into the GCMS (model: QP 2010S). The injector temperature was set at 260°C, and the column (ELITE-5MS: 30 m × 0.25 mm i. d.) temperature was initially set at 80°C. Throughout the procedure, the temperature flow was set to rise at a rate of 10°C/min. Pressure: 65.0 kPa, column flow: 1.00 ml/min, total flow: 24.0 ml/min, purge flow: 3.0 ml/min, and linear velocity: 36.8 cm/s were the requirements. The process ran for 6 min at a final temperature of 280°C.A split injector injected 1 µl of M. zapota extract with some simple ointment base. Slowly, the remaining components of the basic ointment base were added, and everything was thoroughly blended together. The ointment was kept in a cool, dark place away from any heat sources. The ointment was applied topically to the wounds for 20 days in the experiment.White soft paraffin, cetostearyl alcohol, hard paraffin, and wool fat were used to make simple ointments . The 100Wistar rats of both sexes (weighing 190–200 g) were used in all experiments. They were kept in approximately 22 degree Fahrenheit conditions, in polypropylene cages (47 cm × 34 m × 20 cm) lined with husk, which were changed out every 24 h. The rodents could eat and drink freely. The rodents were given a typical pellet diet. With approval from the Najran University Scientific Research Ethical Committee , the institute conducted the animal experiments. These four groups of animals were chosen at random: Group I received no treatment; Group II used povidone-iodine ointment (5% W/W); Group III used MZE ointment (5% W/W); and Group IV used MZE ointment (10% W/W).The standardized extract was studied to identify the appropriate therapeutic dose. On rats with shaved backs, the extract was tested for acute cutaneous toxicity at a 10% (w/w) concentration by applying ointment with the highest concentration of extract. Animals were observed immediately after dosing at least once during the first 30 min, periodically during the first 24 h, with special attention given during the first 2–6 h after the beginning of the exposure period, and daily thereafter, for a total of 14 days. Observations included changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. The research was conducted in accordance with OECD guideline No. 402 .2 and 2 mm in depth was prepared on the shaved dorsal thoracic region (2 as 100%). The epithelization period was determined by counting the days until no more raw wound could be seen after the dead tissue had fallen.The wound was created in anesthetized test animals in accordance with the excision wound model. Ketamine (80 mg/kg) and diazepam (5 mg/kg i. p.) were used as anesthetics to induce anesthesia in the rats before inducing the wound injury. A full-thickness circular excision wound measuring 314 mmc region . Then, ac region . Using tc region , the perOn day 20 of the experiment, the hydroxyproline content of the excised wound tissues was assessed. The tissue samples were dried in a hot air oven operating at 60°C. Following this, they underwent hydrolysis for a duration of 4 hours at a temperature of 130°C in the presence of 6 N hydrochloric acid. The hydrolysates underwent neutralization to achieve a pH of 7.0, followed by oxidation with Chloramine-T for a duration of 20 min. The reaction was terminated at the 5-min mark through the introduction of 0.4 M perchloric acid, followed by colour development using Ehrlich’s reagent at a temperature of 60°C. The specimens were analyzed using an ultraviolet spectrophotometer at a wavelength of 557 nm following thorough agitation. The concentration of hydroxyproline in the tissue samples was ascertained by employing a standard curve of pure L-hydroxyproline .2CO3 in 0.1 N NaOH and 2 ml of 0.5 percent CuSO4 in 1% sodium potassium tartrate, in a distinct container. The samples were subjected to incubation for a duration of 15 min under normal room temperature. A volume of 100 ml of Folin-Ciocalteau reagent was added to each sample and allowed to stand at room temperature for a duration of 30 min. Absorbance at 670 nm was measured within a 30-min time frame using a UV-Vis spectrophotometer  on the final day of the wound healing experiment before being put to sleep. Samples of the wound tissue and the nearby healthy tissue were then taken. The collected samples underwent a standard histopathological tissue examination after being fixed in 10% formalin. Hematoxylin-eosin was used to stain the sectioned wound tissue specimen. A light microscope was used to examine the prepared tissue slide. The wound tissue specimen was cut into sections, stained with a collagen fiber-specific dye called Van-Gieson stain, and then examined under a microscope to determine its collagen content.Dorsal fur was removed from the rats, and a 3 cm long longitudinal paravertebral incision wound was made and sutured 1 cm apart using surgical thread (no. 000) and a curved needle (no. 11), just as in the excision wound model . The thr2 for 24 h to reach 100% confluence as a monolayer. The monolayer over the well’s center was gently scratched with a new 200 µl pipette tip. After gently scratching the well, wash it twice with medium to remove the detached cells. After two washes in 1x Phosphate Buffered Saline, you can refill the well with new media (PBS). Somehow, the PBS went missing. To each well, 1 ml of fresh medium and 25 µl of a range of test concentrations drawn from our stock of test medications were added. After incubating the plate for 24 h at 37°C and 5% CO2, images of scratched monolayers were taken at 0, 12, and 24 h intervals. MagVision Software was used to measure the gap distance at 4X resolution Wi—Initial Wound width (µm)Wt—Final Wound width (µm)T—Duration of migration (hour)Rate of Migration was calculated using below formula:Candida albicans strains were seeded with a glass rod into Petri plates containing 25 ml of optimized media; to evaluate antibacterial activity, Escherichia coli (Gram + ve) and Bacillus cereus (Gram -ve) strains were used. The wells were drilled using a well-borer and the spread plate method. Standard drug (itraconazole and ciprofloxacin) was used in a concentration of 30 µl, and the stock concentration was 100 mg in 1 ml. After that, the plates were left in an incubator at 37°C for a full day. Confirmation of the well’s antimicrobial efficacy was obtained by measuring the diameter of the inhibition zone  was added. The plates were then incubated for 24 h at 37°C. The diameter of the inhibition zone formed around the well was measured to confirm antifungal activity (Petri dishes with 25 ml capacity optimized media were seeded with 24 h old activity .2O2 solution in phosphate buffer (pH-7.4). After 10 min, the solution’s absorbance at 230 nm was measured in comparison to a blank solution made of phosphate buffer without hydrogen peroxide. Phosphate buffer was used in place of the sample or standard to create the control. Triplicate samples of each sample were analyzed. Utilizing the formula method, the percentage of inhibition was calculated.Based on their capacity to scavenge hydrogen peroxide, plant extracts were evaluated for their antioxidant activity using ascorbic acid as a reference . 0.5 ml Where A1 is the absorbance of the control reaction and A2 is the absorbance in the presence of the samples or standards.Using the techniques described by 50 value was determined. Higher levels of free radical activity were indicated by the reaction mixture’s lower absorbance.Where, At is the absorbance of the test sample and Ac is the absorbance of the control reaction (100 µl of ethanol plus 100 µl of DPPH solution). The experiment was carried out three times. For each of the used samples, the ICpost hoc test was done to statistically analyze the data, and all values are presented as the mean and standard error of the mean. Statistical differences were considered significant for p < 0.05.One-way analysis of variance followed by Tukey’s Preliminary phytochemical screening of the ethanolic extract of MZE showed the presence of carbohydrates, flavonoids, steroids, anthraquinone glycosides, tannins and phenolic compounds. The quantification results showed that the extract exhibited phenolic (4577.11 µg GAE/g) of dw content and flavonoid (3603.63 µg QE/g dw).Results of GCMS revealed the presence of 15 major compounds, i.e., Neophytadiene, Tetramethyl-2-hexadecen-1-ol, e-Phytol, Hexadecanoic acid methyl ester, 9,12-octadecadienoic acid -, methyl, 9,12,15-Octadecatrienoic acid, methyl ester, Phytol, Octadecanoic acid, methyl ester, 3-Cyclopentylpropionic acid, 2-dimethylaminoethyl ester, 1-propanone, 1,2-benzenedicarboxylic acid, Threnone, tetramethyl-1,2,3,3a,4,6,8,9,10,10a,11,12,12a,12b-tetradecahydro-benzo, n-Tetracosanol-1, Vitamin E. These results are presented in −1indicates presence of -OH, Phenol, and Alcohol. The complete FTIR results depicting the characteristic peaks, types of chemical bonds and probable functional groups is presented in TheIR spectrum shows peaks at 3315 cmIn acute dermal toxicity, the test extract was safe at a dose of 2,000 mg/kg (limit dose); there was no sign of skin reaction, inflammation, erythema, irritation or redness, and any adverse reaction when the animals were observed for 14 days.M. zapota test extract and Povidone-Iodine were depicted in p < 0.01) wound contraction was observed in animals treated with 10% of test extract and Povidone-Iodine on day 4 as compared to untreated animals (Excision control). On day 8, 10% of test extract and Povidone-Iodine have shown significant (p < 0.001) wound recovery. Whereas, animals treated with M. zapota test extract (5% and 10%) and Povidone-Iodine on day 12, 16 and 20 nearly complete healing of the wound (p < 0.001) (Percentage wound contraction for < 0.001) .p < 0.01) than in the control group who received no treatment. MZE ointment’s wound healing effect was similar to the standard ointment (8 days)  .p < 0.01) higher (34.37 ± 2.7) compared to excision control animals. Furthermore, rats treated with MZE 10% of test extract (66.34 ± 1.7) and Povidone-Iodine (84.33 ± 1.9) have showed significant (p < 0.001) increase in hydroxyproline content as compared to excision control animals.The effect of MZE on Hydroxyproline content in the healed tissue showed in p < 0.001) increased in the animals applied with MZE 10% (83.80 ± 3.6) and Povidone-Iodine (112.6 ± 3.5) compared to excision control group. The total protein content in skin tissue is depicted in p < 0.01) increase in the level of protein compared to excision control group.The protein content in the skin was significantly (p < 0.001) in animals treated with MZE 10% (1067 ± 21.72) and Povidone-Iodine (1133 ± 24.13) compared to incision control animals. While animals treated with MZE 5% (820.0 ± 4.764) showed a significant (p < 0.01) increase in tensile strength as well  and Escherichia coli (Gram-negative). The MZE at 150 µl showed a maximum 13 and 18 mm zone of inhibition for Staphylococcus aureus and Escherichia coli, respectively. Whereas the standard drug Ciprofloxacin has shown 28 mm (Staphylococcus aureus) and 30 mm (Escherichia coli) zone of inhibition. The antifungal activity of MZE observed a zone of inhibition of 7, 9 and 12 mm for 50, 100 and 150 µl for MZE, respectively. Whereas, Itraconazole showed a greater zone of inhibition of 13 mm.The results of antibacterial and antifungal activity are depicted in Hydrogen peroxide radical scavenging assay revealed that plant extract showed appreciable scavenging activity with inhibition percentage 54.44% ± 0.213% which was less compared to standard, i.e. 74.46 ± 0.13 .The DPPH scavenging is measured by decreasing in absorbance by the action of the test extract. The antioxidant activity of plant extract was compared with ascorbic acid as standard antioxidant. The results revealed that the plant extract was similar compared exhibited potent antioxidant activity with a value of 74.82 ± 0.46, which was similar to the standard ascorbic acid with a value of 83.91 ± 0.35 .In the current study, the main aim was to investigate if applying MZE ointment to rats accelerated the process of wound healing. This study’s findings revealed that excision and incision control animals have slowed wound healing. Additionally, the MZE treatment significantly sped up the wound healing rate and decreased the time needed to heal wounds. These findings demonstrate that MZE sped up the rate at which rats’ wounds healed.M. zapota ethanolic extract was determined by using the acute dermal toxicity assay. The extract was found to be safe at a dose of 2000 mg/kg; after 14 days of observation, the animals showed no signs of skin reaction, inflammation, erythema, irritation, or redness. In addition, a previous study evaluated the M. zapota bark extract for cytotoxicity against a normal cell line, human diploid lung fibroblast (WI-38)  , it was Curcuma longa , and the downregulation of nitric oxide and reactive oxygen species (ROS). They also improve the tissue’s antioxidant capacity during the inflammatory phase, increase matrix metalloproteinase and endothelial cell proliferation during the re-epithelialization phase, and enhance the proliferation of damaged tissue cells during the granulation phase, all of which shorten the time needed for a wound to heal .The current study’s preliminary phytochemical analysis of the ethanolic extract revealed the presence of carbohydrates, flavonoids, steroids, anthraquinone glycosides, tannins, and phenolic compounds. This finding was consistent with earlier literature, which also revealed the presence of anthraquinone glycoside, amino acids, proteins, saponin, gums, reducing sugars, tannins, and flavonoids .In the present study, GCMS analysis revealed the presence of 15 major compounds. Some of these compounds were reported to have anti-inflammatory, cytotoxic, antiallergic and antioxidant activities and may be responsible for the antimicrobial and wound healing activity. Neophytadiene , Phytol M. zapota are primarily responsible for its antioxidant capacity. The H2O2 assay is a standard method for assessing the antioxidant capacity of the extracts. Despite being chemically inert, hydrogen peroxide is poisonous to cells because it can damage them by creating hydroxyl radicals. Antioxidants like phenols, polyphenols, and flavonoids can shield human cells from oxidative damage, which can scavenge hydrogen peroxide. The DPPH assay is most frequently used to evaluate the antioxidant activity of various plant-based drugs and crude extracts. It is based on eliminating the coloured free radical DPPH by free radical scavengers in a methanolic solution. With the absorbance at 517 nm, the concentration of free radical scavengers is inversely correlated with DPPH scavenging. The M. zapota extract in the current study demonstrated strong H2O2 and DPPH scavenging activity.Reactive oxygen species and oxidative stress are required in small amounts for normal wound healing, but too much of either hinders the process. By reducing the oxidative stress that wounds cause, antioxidants are thought to hasten wound healing . The polM. zapota leaf extract as a natural antimicrobial agent has been examined in several studies. According to a study, the stem bark and leaf ethyl acetate extracts showed variable antimicrobial activity against bacteria and fungi. It has been demonstrated that several phytochemicals, including terpenoids, flavonoids, and glycosides, have antimicrobial properties. Thus medicinal plants offer a novel source of antibacterial and antifungal agents that exhibit significant efficacy against pathogenic microorganisms. In the present investigation, the antimicrobial activity of the plant extract was found to be more effective against Gram-negative bacteria than Gram-positive bacteria. The potential antioxidant and antimicrobial properties of M. zapota’s active phytoconstituents are responsible for the improved wound healing seen in the current study  or mechanical fixation of the skin using specific tools or splints are two strategies to combat this condition. Using reliable wound evaluation techniques will aid in understanding the mechanics of wound healing and maximize efforts into potentially beneficial therapies that can accelerate the healing process.M. zapota ointment has great potential for the future of wound healing and also being cost-effective at the same time.Chronic, non-healing wounds place a heavy financial burden on the healthcare system, significantly decrease the quality of life for those affected, and frequently forecast more terrible outcomes such as limp amputations or even early deaths. An aging population and the rising incidence of lifestyle disorders like obesity and diabetes will only add to this problem. Drugs for wound healing should ideally be developed to reduce patient morbidity and suffering, be effective and rapid in providing outcomes, and, most significantly, be cost-efficient.


Curcuma longa L.)—NOMICU® L-100 (N) in the formulation of probiotic yogurt in comparison with the standard turmeric extract (TE). The antimicrobial activity of both supplements was studied and compared in the context of gram-positive and gram-negative bacteria, yeasts, and fungi. The N maintains the level of Bifidobacterium animalis subsp. lactis BB-2 in yogurt at the recommended level (7–9 log CFU/g) throughout the storage period. NOMICU® L-100 also has a higher inhibitory capacity for the growth of yeast and fungi. The evaluation of quality indicators of yogurt with N and TE at the level of 0.2% proves that yogurt with N has original taste properties. A lower degree of syneresis was noted for yogurt with TE (0.2%), but its sensory properties are unacceptable to the consumer due to the appearance of a bitter taste. In conclusion, based on the obtained results, it has been proven that the use of NOMICU® L-100 (0.2%) in the composition of yogurt provides a product of functional direction with stable quality and safety indicators, which can be stored for at least 28 days.Curcumin is a nutraceutical with unique anti-inflammatory, anti-oxidative, and antimicrobial properties. In this study, we aimed to examine the advantages of the use of water dispersible and highly bioavailable form of standardized turmeric extract ( Fermented milk drinks are classified into three types based on their shelf life: fresh fermented milk drinks with a short shelf life, those with an extended shelf life, and thermalized fermented milk drinks , 2. At tFor the mass production of fermented milk products, it is also common to use preservatives, which stop the decomposition processes that occur in non-living cells and reduce the activity of metabolic processes in living cells, thereby ensuring the product’s long-term storage , 14. In Aspergillus spp., A. parasiticus, and Penicillium camemberti are inhibited by a concentration of 500 mg/kg of sorbate. Furthermore, adding potassium sorbate to yogurt at concentrations of 0.5%, 0.1%, and 0.2% inhibited yeast and fungi levels, with normal characteristic properties extending more than 14 days . The antibacterial activity of various probiotic strains plays an important role in increasing the shelf life of various types of food products. Their combination with a bioavailable form of curcumin, innovative packaging methods, and the selection of raw materials with a high degree of microbiological purity can ensure the production of a prophylactic fermented milk drink with an extended shelf life without the use of traditional artificially synthesized additives that have a negative effect on the human body.The complex of the bioactive form of curcumin and milk proteins is able not only to provide preventive direction to the fermented milk drink but also to show a synergistic effect together with lactic acid bacteria, thereby increasing their vital activity . Thus, Aorescens . Importaorescens . It was Bifidobacterium animalis subsp. lactis BB-12, and to suppress the development of coliforms, yeasts, and fungi in order to select the best doses of curcumin preparations  in yogurt formulations for further research, and finally to investigate the effect of selected doses of turmeric extract and NOMICU® L-100 on the physicochemical indicators of yogurt , as well as to evaluate sensory indicators of the experimental samples during the storage period of 28 days.Thus, we aimed to investigate the effect of the bioavailable form of curcumin on the viability of probiotic bacteria, antibacterial activity against pathogenic microorganisms, and quality indicators of yogurt. Specifically, our purpose was to determine and compare the antimicrobial activity of selected curcumin preparations , to test the hypothesis regarding the ability of curcumin to influence the viability of probiotic cultures using the example of 2.2.1.Curcuma longa L. (TE) with additives, permitted in food supplements (compliant with regulation (EС) № 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives, GRAS—Generally Regarded as Safe). The N is a proprietary formulation containing a full spectrum of curcuminoids for use as an active ingredient in dietary and food supplements, functional beverages and dairy products, cosmetics, and MCT oils. The second one is TE, which contains 95% of curcumin. For yogurt production, the culture starters YC-X16  and BB-12  were used.For the experiment, two forms of curcumin were used. The first, NOMICU® L-100 (N), is a water-dispersible, highly purified, and highly bioavailable form of standardized turmeric extract 2.2.Curcuma longa L.)—NOMICU® L-100. Next, the milk was inoculated with a single starter culture YC-X16  and Bifidobacterium animalis subsp. lactis BB-12 . An activated starter was made by inoculation of milk with DVS cultures. Each batch of milk was inoculated with a previously activated starter culture (in the form of bulk activated at 40°C for 10 h). Inoculated milk was stirred and fermented at 40°C for 10 h. After fermentation, curcumin in different forms (TE and N) was added in amounts of 0%, 0.10%, 0.15%, 0.20%, and 0.25%, respectively. The final products were stirred and poured into 100-ml plastic cups and cooled to 5°C.The milk with 3.2% of fat with skimmed milk powder of 1.5% was pasteurized at 85°C for 30 min, then cooled to 40°C, and divided into groups including C—control milk, TE—sample with turmeric extract addition of curcumin form; N—turmeric extract  and gram-negative  bacteria, yeasts , fungi  were used as references for the antimicrobial assay of N and TE. Bacteria and yeasts were purchased from the Czech Collection of Microorganisms . After 24 h, single colonies on agar plates were used to prepare the microbial suspension with a turbidity of 0.5 McFarland [equal to 1.5 × 108 colony-forming units (CFU/mL)]. The turbidity of the bacterial suspension was measured at 600 nm. MHA agar plates were inoculated with bacterial strain under aseptic conditions, and wells (d = 6 mm) were filled with 50 μL of the test samples and incubated at 37°C for 24 h. Microscopic fungi and yeasts were evaluated on SDA at 25°C for 24 h. After the incubation period, the diameter of the growth inhibition zones was measured in triplicate.For antimicrobial analyses of clear powder, the well agar diffusion method was used. Antibacterial activities of N and TE were evaluated using the well diffusion method on Mueller-Hinton agar  for bacteria and Sabouraud Dextrose Agar  for yeast and microscopic fungi. The inhibition zones were reported in millimeters (mm). Gram-positive (GThe Minimum Inhibitory Concentrations (MIC) of bacteria and yeasts were determined using the agar microdilution method. The inoculum was cultured for 24 h in Mueller-Hinton Broth  at 37°C for bacteria and Sabouraud Dextrose Broth  at 25°C for yeast. A sample of 100 μL of nutrient medium and 50 μL of inoculum with an optical density of 0.5 McFarland standard were applied to a 96-well microtiter plate. Subsequently, N and TE were prepared by serial dilution to a concentration range of 11 mg/ml to 0.010 mg/ml in MHB / SDB and mixed thoroughly with bacterial and yeast inoculum in the wells. The prepared 96-well microtiter plates were measured at 570 nm with a Glomax spectrophotometer  at 0 h. Subsequently, the bacterial samples were incubated at 37°C for 24 h. Yeast samples were incubated at 25°C for 24 h and measured again. MHB/SDB with essential oil was used as a negative control, and MHB/SDB with inoculum was used as a positive control for maximal growth.2.3.2.Streptococcus and MRS for Lacticaseibacillus and Bifidobacterium . Lacticaseibacillus and Bifidobacterium were cultivated in microaerophilic conditions with 5% CO2 at 37°C. The identification of Streptococcus and Bifidobacterium was performed using MALDI-TOF MS.To conduct the study, 5 g of each sample of yogurt was diluted in 45 ml of physiological solution. The microbiological analysis was carried out at the following conditions: total number of microorganisms on PCA  at 30°C for 48–72 h, number of coliforms bacteria on VRBL agar  at 37°C for 24–48 h, number of lactic acid bacteria on MRS , MSE  agars at 37°C for 48–72 h, and microscopic fungi and yeasts on MEA  at 25°C for 5 days. The studies were carried out using the plate method with M17 agar  for 2.4.2.4.1.−1.The pH value in milk after fermentation was determined by pH-meter FiveEasy  using InLab®Solids Pro-ISM electrode . Lactic acid content was determined according to Jemaa et al. . Ferment2.4.2.The color was determined by a colorimeter  using the CIE LAB system. The following parameters were determined: L*—as lightness (from 0—black to 100—white), a*—as color from red (+) to green (−), b*—as color from yellow (+) to blue (−), C—as color purity and intensity, and h—as color hue. Before measurement, the device was calibrated on a white reference standard.2.4.3.Syneresis was determined by the centrifuge method using the Laboratory Refrigeration Centrifuge LMC-4200R  according to Lopez-Santamarina et al.  method w2.4.4.The stability of the aqueous dispersion of curcumin was measured in the sedimentation assay using a centrifuge MPW 351R with rotor no. 12177. TE and N were dispersed in 50 ml of distilled water and centrifuged in falcon tubes at 226 g for 10 min, at 19 ± 1 °C. Absorbance of the supernatant was measured at 450 nm on a spectrophotometer, .Sedimentation (%) was calculated using the formula:2.4.5.The sensory evaluation was carried out by a trained panel of experts for probiotic yogurt with curcumin  and without curcumin at the 7th, 14th, 21st, and 28th days of refrigerated storage. Parameters were evaluated on a 9-point scale (from 1 = undetectable to 9 = very intense). The following parameters were evaluated: milky–creamy taste, sour taste, sweet taste, the taste of the additive, off-taste, fermentation odor, odor of the additive, and off-odor.2.4.6.p ≤ 0.05. Data are expressed as mean with standard deviations (±SD), and differences between groups were assessed using Tukey’s test. All the analysis was done in triplicate.The analysis of variance (ANOVA) was performed using STATISTICA 13 software. Test significance was set at 3.3.1.+ and G− bacteria, yeasts, and mycelial fungi. TE and N show different antimicrobial activity against the studied microorganisms (+ bacteria with an inhibition zone of 8.00 ± 0.02 mm (S. pneumoniae), G− bacteria with an inhibition zone of 3.00 ± 0.02 and 5.00 ± 0.02 mm , and fungi with an inhibition zone of 7.00 ± 0.02 and 6.00 ± 0.04 mm . At the same time, for TE, the highest antimicrobial activity was observed against G+ bacteria with an inhibition zone of 8.00 ± 0.02 and 9.00 ± 0.01 mm , G bacteria with an inhibition zone of 2.00 ± 0.01 mm (E. coli), yeast with an inhibition zone from 5.00 ± 0.02 mm  to 7.00 ± 0.02 mm , and microscopic mycelial fungi from 6.00 ± 0.03 mm  to 9.00 ± 0.01 mm (P. chrysogenum). The conducted MIC test for N revealed the lowest value for MIC 50 (0.09 mg/ml) for C. tropicalis and G+ bacterium (S. pneumoniae) and the highest for MIC 50 (3.60 mg/ml) and MIC 90 (4.20 mg/ml) for G+ bacterium (S. aureus). For TE, the test showed the lowest value of MIC 50 (0.06 mg/ml) and MIC 90 (0.09 mg/ml) for the G+ bacterium (Streptococcus pneumoniae) and the highest for MIC 50 (3.60 mg/ml) and MIC 90 (4.20 mg/ml) for G+ bacteria (S. aureus).The analysis of the antimicrobial activity of TE and N was carried out using Grganisms . Thus, N3.2.Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), b—Lactobacillus acidophilus LA-5® , c—Lacticaseibacillus casei 431® , d—Lactobacillus johnsonii La 1 , e—Bifidobacterium animalis subsp. lactis BB-12  are shown in , Lactobacillus acidophilus, Lactobacillus johnsonii, and Lacticaseibacillus casei. At the same time, for Bifidobacterium animalis subsp. lactis BB-12, the same effect is observed for both supplements (N and TE). For the yogurt cultures, the activity of N (0.15%–0.25%) is observed from the first hours, whereas for TE (0.15%–0.25%), the active growth of this strain begins after the 6th hour and reaches an insignificantly smaller number of microorganisms in comparison to N (log CFU/g 8.34–5.53). For Lactobacillus acidophilus, the growth of bacteria in the case of N (0.15%–0.25%) is dynamic, while for TE (0.1%–0.25%), there is a sharp increase in the number of viable cells of the probiotic within the first 6 h, leading to a plateau, where the number of probiotic bacteria does not change in a significant manner within the next 18 h. The dynamics of the growth of Lacticaseibacillus casei microorganisms are somewhat different. In Lactobacillus johnsonii microorganisms, and for TE, on the contrary, it is slightly increased. Despite this, the total number of probiotic microorganisms is the highest for N (8.75 log CFU/g) at doses of 0.1%. The effect of TE and N on Bifidobacterium animalis subsp. lactis BB-12  is marked by stable dynamic growth. TE (0.1%) within 18–20 h shows the most active effect on probiotic microorganisms, which then decreases slightly and gives way to N (0.1%–0.15%).The results of growth dynamics of bacteria cultures: a—yogurt cultures   . On the g CFU/g) . At the g CFU/g)  than in r strain . Althougr strain .3.3.Active acidity for both TE and N samples was slightly lower than in the control sample . Thus, iThe degree of syneresis in experimental yogurt samples increased during 28 days of storage: for the control sample—from 60.01% ± 0.40 to 63.16% ± 0.38; for TE—from 61.66% ± 0.12 to 62.34% ± 0.07 and for N—from 61.94% ± 0.82 to 65.11% ± 0.07. The addition of TE insignificantly reduced the degree of syneresis, while N increased it, which is related to its effect on the stratification of the milk clot.The results of the chrominance study indicated that the control sample had a higher brightness than the other yogurt samples and a green color . The addition of TE reduces the intensity of the green color, and N shifts it toward the red . All samples have a yellow color. Saturation and color stability were the lowest for the control sample and amounted to 9.81 ± 0.05 units at the end of the storage period. This indicator significantly increased with the addition of curcumin preparations: for TE, the maximum value reached 36.56 ± 0.12, and for N—68.70 ± 0.16. For the h, a slight decrease was observed for the samples TE and N, compared to the control. The results of the measurement of the yellowness index are consistent with the data for (b*). For the control, its decrease was observed during storage, while for TE, it increased from 60.82 ± 0.99 to 62.12 ± 0.11 and for N from 94.45 ± 0.82 to 96.17 ± 0.25.L* is lightness (from 0—222 black to 100—white), a* is color from red (+) to green (−), b* is color from yellow (+) to blue (−), C is color purity, and intensity and h is color hue.Explanatory note: physicochemical indicators of yogurt samples during 28 days of storage, where the following subscript mean: Sensory evaluation of experimental samples of yogurt was carried out on the 7th, 14th, 21st, and 28th days of storage .From the obtained data, it can be seen that the most pronounced sour-milk taste was observed for the control sample, which became more saturated during storage, while the addition of both curcumin preparations slightly reduced its intensity, especially for N during the first 3 weeks, but at the end of the storage period, it was the same for both N and TE, which correlates with the data on the amount of lactic acid formed in the experimental samples. The sour-milk aroma in the case of TE application was greater than that of N during the first 3 weeks, but at the end of the storage period, it was barely perceptible. Thus, for TE, already on the 7th day, the aroma of the additive was observed, which increased during the 28 days of storage, which is not a characteristic feature of yogurt . At the 3.4.As can be seen from 4.4.1.+ bacteria  and GP− bacteria   . The mecaecalis) . Unlike aecalis) . Despite− bacteria (P. aeruginosa and Y. enterocolitica), which might be attributed to a stabilizing effect of the formulation, based on GRAS additives, which to some extent modifies the properties of curcumin, in particular antibacterial ones  log CFU/g, and in cow’s milk at the level of (7.10 ± 0.85) log CFU/g. In our experiment, it was determined that the form of curcumin (NOMICU® L-100) in fermented cow’s milk allows ensuring this indicator on the 28th day of storage at the level of 7.7 log CFU/g, while the results of the effect of TE on the survival of the probiotic during the 21st day are comparable to the generally known scientific data. The synergistic effect of curcumin with probiotic cultures was also noted by other scientists (6 CFU/ml) at the end of the storage period.Natural food additives with nutraceutical properties are attractive to food manufacturers, but their presence in fermented milk products can affect the growth and development of lactic acid bacteria in different ways . Additivientists , who shoThere are also studies available on the use of nanoemulsions of natural functional ingredients to support the development of probiotic cultures and secure the recommended level of 6 log CFU/g during the entire storage period . Sharma L. rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12  (56), what was established during our study. Such a fenomena may be related to the fungicidal effect of curcuminoids, which could suppress the vital activity of such cultures as Streptococcus thermophilus, Lactobacillusdelbrueckii subsp. bulgaricus, YF-L812, Bifidobacterium animalis subsp. lactis BB-12, and others  is known to have a positive effect on the growth of is BB-12 . Howeveris BB-12 . At the d others . Yazdi eelopment . The pos4.3.Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Guerra et al.  may be due to a combination of factors, such as the presence of riboflavin in milk used for the production of yogurt samples , oxidatiSome researchers  suggesteAnother study noted the ability of TE to impart a spicy flavor to yogurt . In thisThe positive effect of encapsulated curcumin in a dairy food system with a low-fat content was noted by various scientists who pointed to its ability, in the absence of fat, to form connections with micelles of milk proteins . Moreove4.4.Szterk et al.  reported4.5.In the last decades, there have been many attempts to enrich different types of food with natural products with beneficial properties . It is e5.+ bacteria (S. pneumoniae) and fungi .1. NOMICU® L-100 formulation (N) shows antimicrobial activity against pathogenic microorganisms, with the highest activity observed against GBifidobacterium animalis subsp. lactis BB-12. For N, the amount of probiotics was at the level recommended for functional fermented milk drinks (7–9 log CFU/g) throughout the entire storage period.2. Based on the conducted microbiological analysis of yogurt samples with TE and N (mass fraction −0.1% to 0.25%) on the 7th, 14th, 21st, and 28th day of storage, it was established that these additives show not only antibacterial activity but also support the development and vital activity of the probiotic culture 3. Evaluation of the quality indicators of yogurt with N (0.2%) indicates that the formation of lactic acid occurs at almost the same level as in the control sample. It should be noted that this additive improves the color indicators of the product, which increases its attractiveness to the consumer. Sensory evaluation of experimental yogurt samples during 28 days of storage confirmed that N (0.2%) provided the best taste and aroma indicators.4. Thus, the use of N (0.2%) due to its antibacterial properties, as well as the ability to support the vital activity of probiotic microorganisms, allows for avoiding the use of other preservatives in the yogurt formulation. Unlike crude turmeric extract (TE), N has excellent properties to enhance the taste of milk fat and give the product an attractive color, which makes the fermented dairy product even more attractive to consumers.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.MB-O and JU: conceptualization. AM and MK: methodology. AZ-P: software. MB-O, MB, and JU: validation and supervision. MB: formal analysis. MB-O, AM, MK, and PB: investigation. AM: resources. MB-O: data curation and visualization. MB-O, AM, JU, and MB: writing of original draft preparation. MB and MK: writing of review and editing. JU: project administration and funding acquisition. All authors contributed to the article and approved the submitted version.The research project was carried out with the support of the funds of the Polish National Center for Research and Development, under the framework program 1.3.1. The technology for extending the shelf life of probiotic-nutraceutical dairy products is protected by patent application P.443720 filed by Dairy Biotechnologies Ltd.Author JU was employed by the company Dairy Biotechnologies Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The wearable cyborg Hybrid Assistive Limb (HAL) is the world’s first cyborg-type wearable robotic device, and it assists the user’s voluntary movements and facilitates muscle activities. However, since the minimum height required for using the HAL is 150 cm, a smaller HAL (2S size) has been newly developed for pediatric use. This study aimed to (1) examine the feasibility and safety of a protocol for treatments with HAL (2S size) in pediatric patients and (2) explore the optimal method for assessing the efficacy of HAL.t-test or Wilcoxon signed-rank test.This clinical study included seven pediatric patients with postural and motor function disorders, who received 8–12 sessions of smaller HAL (2S size) treatment. The primary outcome was the Gross Motor Function Measure-88 (GMFM-88). The secondary outcomes were GMFM-66, 10-m walk test, 2- and 6-min walking distances, Canadian Occupational Performance Measure (COPM), a post-treatment questionnaire, adverse events, and device failures. Statistical analyses were performed using the paired samples p = 0.07). The improvements in the secondary outcomes were as follows: GMFM-66, 53.63 ± 11.94 to 54.96 ± 12.31, p = 0.04; step length, 0.32 ± 0.16 to 0.34 ± 0.16, p = 0.25; 2-MWD, 59.1 ± 57.0 to 62.8 ± 63.3, p = 0.54; COPM performance score, 3.7 ± 2.0 to 5.3 ± 1.9, p = 0.06; COPM satisfaction score, 3.3 ± 2.1 to 5.1 ± 2.1, p = 0.04.All participants completed the study protocol with no serious adverse events. GMFM-88 improved from 65.51 ± 21.97 to 66.72 ± 22.28 , to children with central nervous system disorders. We evaluated its safety, feasibility, and identified an optimal assessment method for multiple treatments. All participants completed the protocol with no serious adverse events. This study suggested that the GMFM would be an optimal assessment tool for validation trials of HAL (2S size) treatment in pediatric patients with posture and motor function disorders. FurtherThe wearable cyborg Hybrid Assistive Limb®  is the world’s first cyborg-type wearable robotic device , 7. The Consequently, a new smaller-sized HAL  was developed for use in patients with postural and motor function disorders. Nakagawa et al. reported an immediate effect and safety profile of single-session treatment with the smaller HAL, and confirmed significant improvements in walking speed, step length, and joint angles of the lower limbs . In addi2.2.1.We conducted a single-arm study to compare the effects of HAL (2S size) before and after treatment in children with postural and motor function disorders, including CP, spina bifida, cerebrovascular disorders, and cerebrospinal cord injuries. Because this study was designed as an exploratory study to test the efficacy of HAL in pediatric patients with brain and spinal cord disorders, patients with multiple disorders were included. The inclusion criteria were as follows: (1) written consent provided by their substitute; (2) aged 5–15 years; (3) classified as level II–IV on the gross motor function classification system (GMFCS); (4) at least 1 year passed since the disease onset; (5) compatible with the HAL (2S size) device; and (6) willing for continuous hospitalization or outpatient visits according to the study schedule during the study period. The GMFCS classifies the severity of gross motor function impairment in children with disabilities . The GMFSubjects were recruited at each site where the study was conducted, and consent was obtained from both family members and subjects using an explanatory document.2.2.In this study, HAL (2S size) was used . The conThe HAL (2S size) treatment was performed once a day for 20 min for a total of 8–12 sessions, during a 5–8 weeks intervention period. It consisted of knee flexion/extension training, stand-up training, standing, and gait training; warm-up and cool-down were performed before and after treatment, respectively, as well as the outcome measures. Assessments at the beginning were made 1 week before the treatment period and assessments at the end were made 1 week after the end of treatment. This study was conducted from July 2020 to July 2021 at the University of Tsukuba Hospital, Ibaraki Prefectural University of Health Sciences Hospital, and the National Hospital Organization Niigata National Hospital. This study was conducted in an outpatient or inpatient setting. Patients who lived too far from the study location or were unable to participate as outpatients were admitted to take part in the study.2.3.The primary outcome measure was GMFM-88 score, the gold-standard assessment of gross motor function in children. The evaluation items covered five fields: (A) lying down and rolling; (B) sitting; (C) crawling on hands and knees, and kneeling; (D) standing; and (E) walking, running, and jumping . The GMFThe GMFM-66 is an interval scale version of the GMFM-88, an ordinal scale, reduced to 66 items by Rasch analysis for easy evaluation. By inputting the results of the 66 items into a dedicated program, the GMFM-66 score is calculated; this indicates the gross motor function of children with CP in relation to a 5-year-old child with typical development set at 100 . The COP2.4.p < 0.05 was considered statistically significant with a confidence coefficient of 95%. All analyses were performed using R software.Statistical analyses were performed using paired samples t-test or Wilcoxon signed-rank test, depending on the distribution of the data. Two-sided The study protocol was designed according to the Declaration of Helsinki and relevant ethical guidelines for clinical research. This study was approved by the Tsukuba University Clinical Research Review Board (TCRB19-025) and the Ibaraki Prefectural University of Health Sciences (e261). The study protocol was approved by the Japan Registry of Clinical Trials (jRCTs032200037).3.Seven patients participated in and completed the study protocol. No children were excluded because of their inability to train for HAL. There were three boys and four girls between the age of 6–10 years , with mean height 120.1 ± 7.8 cm  and weight of 25.9 ± 8.8 kg . Five were diagnosed with CP and two with spinal cord disorders. The severity of mobility was GMFCS level II in two patients, III in two, and IV in three. Three patients participated in the study as outpatients, whereas the other four were hospitalized. The details of the patients are presented in p = 0.07. The changes in secondary outcome measures after the treatment were as follows: GMFM-66, 53.63 ± 11.94 to 54.96 ± 12.31, p = 0.04; step length, 0.32 ± 0.16 to 0.34 ± 0.16, p = 0.25; 2-MWD, 59.1 ± 57.0 to 62.8 ± 63.3, p = 0.54; COPM performance score, 3.7 ± 2.0 to 5.3 ± 1.9, p = 0.06; COPM satisfaction score, 3.3 ± 2.1 to 5.1 ± 2.1, p = 0.04  , GMFM dip = 0.12 . The samp = 0.12 .p = 0.008, satisfaction p = 0.012; Owing to the nature of COPM, which extracts subjective work goals from patients, evaluators should not intentionally guide goal setting. Therefore, the results of the original COPM showed that there were many goals that may not relate to HAL (2S size) treatment. Thus, we performed a secondary analysis with the goals only related to Wilcoxon’s signed rank-test . COPM peFurthermore, the questionnaires indicated a high level of satisfaction with a score of 9.1 for the question “Are you satisfied with HAL (2S size-prototype)?” and 9.0 for “Are you satisfied with your HAL (2S size) treatment?.” Participant’s parents had scores of 9.1 and 9.3, respectively, for the same questions indicating that they were also highly satisfied . Additio4.This study was conducted to examine the feasibility and safety of HAL (size 2S) treatment and to identify the optimal assessment index. The absence of serious adverse events was comparable to previous studies that evaluated the safety of the HAL (2S size) in pediatric patients with postural and motor function disorders , 14. We The primary outcome measure, the GMFM-88, did not show any statistically significant improvements.In recent years, the Minimal Clinically Important Difference (MCID) has gained attention as a standard method for measuring clinical appropriateness and to determine the quality of treatment . The MCIIn terms of feasibility, this study included only a small number of patients (n = 7), involving three children with severe disorders with GMFCS level IV, which made it difficult to improve the GMFM . SubjectRegarding walking ability, the results of the 10-MWT, 2-MWD, and 6-MWD improved from the first to the final assessment, but the differences were not significant. Thompson et al. reported that the intra-class correlation coefficient for the 10-MWT for children with CP ranged from 0.58 to 0.78 and the minimum detectable change ranged from 1.7 to 12.2 s, an assessment with a large range of measurement error . For othFurthermore, an increase in COPM satisfaction was observed. In pediatric robot-assisted gait training studies, it is important to reflect on the rehabilitation goals of individual children in the assessment measures. COPM is a valid assessment tool ; we founThis study has a few limitations. First, it was conducted using a single-arm design with patients evaluated before and after the intervention and without a control condition, making it susceptible to confounding factors. Second, the statistical power was low due to the small sample size. Nevertheless, this study was intended as a preliminary study for larger studies to be conducted in the future. The results of this study will be used to develop a protocol for a randomized controlled trial with the presence of a control group.5.In this study, we applied a new size of the wearable cyborg HAL (2S size) to children with central nervous system disorders, and examined its safety, feasibility, and optimal assessment for multiple treatments. All participants completed the protocol with no serious adverse events. Additionally, the GMFM was determined to be the optimal assessment tool to evaluate the HAL treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Tsukuba University Clinical Research Review Board and the Ibaraki Prefectural University of Health Sciences. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Written informed consent was obtained from the individual(s) for the publication of any identifiable images or data included in this article.KTa: Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft. MM: Conceptualization, Funding acquisition, Investigation, Methodology, Writing – review & editing. HW: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Validation, Writing – review & editing. MK: Conceptualization, Investigation, Methodology, Writing – review & editing. YSh: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Writing – review & editing. TaN: Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Writing – review & editing. HM: Resources, Writing – review & editing, Funding acquisition, Investigation. HKam: Conceptualization, Funding acquisition, Writing – review & editing. KTo: Investigation, Writing – review & editing. YH: Conceptualization, Funding acquisition, Resources, Writing – review & editing. KK: Investigation, Methodology, Validation, Writing – review & editing. KY: Investigation, Methodology, Writing – review & editing. ToN: Investigation, Resources, Writing – review & editing. NI: Conceptualization, Funding acquisition, Investigation, Resources, Writing – review & editing. HKaw: Conceptualization, Funding acquisition, Writing – review & editing. YSa: Conceptualization, Funding acquisition, Writing – review & editing. MY: Conceptualization, Funding acquisition, Writing – review & editing. AkM: Conceptualization, Funding acquisition, Writing – review & editing. AiM: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing.


P < 0.05). In the VRR, 13 glands of SHPT (72.2%) and 42 glands of PHPT (87.5%) had achieved the clinical success. In addition, the preoperative and postoperative serum iPTH were higher in the SHPT group than in the PHPT group (P < 0.05). In terms of the serum iPTH and calcium, the PHPT group had substantially higher rates of clinical success, with 42 patients (87.5%) and 46 patients (95.8%) meeting the criteria, respectively compared to 3 patients (33.3%) and 6 patients (66.7%) of SHPT group (P < 0.05). After RFA, the clinical symptoms improved in both groups. The overall incidence of complications (hoarseness and postoperative hematoma) of RFA in the two groups was 10.5% (6/57), and hoarseness  of SHPT group was more common than PHPT group. All the complications were resolved spontaneously within 12 months after symptomatic treatments. In the treatment of PHPT and SHPT, ultrasound-guided RFA is both successful and safe. PHPT patients have better results in restoring normal iPTH by RFA, and have no considerable difference with the SHPT patients in terms of serum calcium, the volume of the ablation area, and the VRR.There is now growing interest in the use of Ultrasound-guided radiofrequency ablation (RFA) to treat hyperparathyroidism. But the efficacy and limitations of this treatment have not been described in sufficient detail. Assessing and contrasting the effectiveness and safety of RFA in treating primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism (SHPT). This retrospective study included 57 HPT patients (48 for PHPT and 9 for SHPT) who underwent RFA between January 2017 and April 2021. The serum intact parathyroid hormone (iPTH) and calcium, hyperplastic parathyroid volume, volume reduction rate (VRR) before and after RFA, clinical success rate, symptoms, and complications were analyzed and compared. In SHPT group, bone pain , skin pruritus , and multiple hyperplastic parathyroid glands  were more common compared to the PHPT group. After 12 months of follow-up, the serum iPTH, calcium, and the volume of PHPT and SHPT groups had decreased by more than 60%, 10%, and 90%, respectively ( Hyperplastic parathyroid glands are often responsible for PHPT, an endocrine condition typified by excessive intact parathyroid hormone (iPTH) and hypercalcemia. Common clinical manifestations of PHPT include bone pain, osteoporosis, kidney stones, skin itching, gastrointestinal symptoms, and neuropsychological symptoms3. One of the most serious side effects of end-stage renal disease is SHPT. It is frequently associated with parathyroid hyperplasia and chronic increases in serum iPTH and calcium. Common clinical manifestations are bone pain, pathological fractures, cardiac arrhythmias, pruritus, and cognitive impairment. Because the end-stage renal disease has been present for a long time, SHPT does not respond well to conventional medical therapy5. As a result, for symptomatic PHPT and SHPT patients who have experienced treatment failure because of medication interactions, resistance, or adverse effects, parathyroidectomy, as a conventional surgical approach, is commonly chosen to normalize serum iPTH and calcium, arrest disease progression, and alleviate clinical symptoms6.The two kinds of hyperparathyroidism that are most frequently encountered are primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism (SHPT)7. In addition, SHPT patients frequently have serious diseases of their heart, lungs, and kidneys, a greater risk of general anesthesia-related postoperative complications and poor tolerance to parathyroidectomy are common in SHPT individuals who require the surgery8. Therefore, patients with PHPT and SHPT need a treatment modality that is less invasive, effective, fast to recover, and aesthetically pleasing. Currently, the surgical effectiveness of benign thyroid nodules treated with minimally invasive procedures like ultrasound-guided (US-guided) radiofrequency ablation (RFA), is equivalent to that of traditional thyroidectomy10. In recent years, US-guided RFA has also been gradually used to treat PHPT and SHPT12. Several literature reports have clearly pointed out that RFA, microwave ablation, and other techniques can validly normalize serum iPTH, calcium, and decrease the volume of glands15. In this study, patients were separated into PHPT and SHPT groups and all were treated with US-guided RFA. Finding out the assessments and contrast of the efficacy and safety after RFA in the two groups were the objectives of this research.However, PHPT patients often suffer from scarring from classic parathyroidectomy, possible postoperative hypocalcemia, recurrent laryngeal nerve (RLN) damage, hypoparathyroidism, as well as continued oral calcium preparations. As a consequence, they refuse to undergo parathyroidectomy resulting in the aggravation of symptoms such as bone pain and osteoporosisThis study involving human participants was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. It was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants and their legal guardians. The research involving human research participants has been performed in accordance with the Declaration of Helsinki. Our study was a retrospective review of 57 HPT patients (48 for PHPT and 9 for SHPT) who consented to undergo RFA from January 2017 to April 2021. The baseline clinical characteristics were analyzed  symptomatic PHPT patients; (2) symptomless PHPT patients who have one of the following conditions: (a) higher serum calcium than the normal range; (b) considerably reduced bone mineral density and a higher probability of suffering a fragility fracture; (c) creatinine clearance < 60 mL/min; (3) SHPT patients who have failed pharmacological treatment; (4) HPT patients with one of the following conditions: (a) at least 1 hyperplastic parathyroid gland found to be amenable to RFA after imaging examination ; (b) unallowed or rejective to have observational regimens or parathyroidectomy; (c) Aspiration biopsy confirmed benign; (5) The Exclusion criteria: (1) known history of neck operation; (2) accompanying a coagulation problem; (3) patients with bleeding disorders, serious infectious diseases, or pregnancy; (4) patients with severe underlying diseases that are ineffective for drug treatment; (5) patients with imaging proof of parathyroid cancer; (6) < 12 months of follow-up.99mTc- MIBI SPECT/CT imaging to locate hyperplastic glands and assess surrounding anatomy: 1. Ultrasound imaging criteria: a well-delimited round, fusiform, or long uniformly hypoechoic masses with complete envelope generally, etc20. 2. 99mTc- MIBI SPECT/CT imaging criteria: (1) In the early phase, there is a concentration of foci with mild to obvious abnormal radioactivity distribution (radioactivity is uniformly distributed in the thyroid). (2) In the delayed phase, there is a concentration of foci with residual or enhanced abnormal radioactivity distribution (regression or mild radioactivity concentration in the thyroid) 20. Laryngoscopy was used before RFA to check if RLN damage already existed. Cardiopulmonary function assessment: In order to determine if the patient would be able to withstand RFA, preoperative electrocardiogram, X-ray examination, computed tomography, and magnetic resonance imaging of the chest were all conducted.Imaging examination  was inserted into the hypoechoic nodules in the hyperplastic parathyroid region, and mobile ablation was performed at a power of 20–40 W. One ablation was performed for approximately 3 min. The RFA was considered complete when hyperechoic microbubbles fully surrounded the nodule. Contrast-enhanced ultrasonography was then performed again to evaluate the microcirculation perfusion of the lesion, and the needle was slowly withdrawn along the cautery needle tract. The entire RFA procedure took approximately 20–25 min. All the hyperplastic parathyroid glands were ablated. During the RFA process, intermittent communication with the patient was used to check for any abnormal sounds or intraoperative pain. Remember to check patients for vital signs after the RFA and observe whether patients have complications such as neck pain, swelling, and dyspnea. Patients were required to be fasting for 4 h, not talk loudly, press their necks for 30 min, and then apply ice-salt packs for 1–2 h to reduce tissue edema and local bleeding.An experienced RFA specialist performed the treatment with routine electrocardiogram monitoring. The patient entirely exposed the neck by lying supinely. After the neck was sterilized, a color ultrasound system (Toshiba Apli0 500 type color ultrasound system) was used to assess again. To assess the glands and blood perfusion, and 2 ml of ultrasound contrast agent  was injected into the cubital vein. Local infiltration anesthesia was performed using 2% lidocaine dilution. Under the guidance of ultrasound, a puncture needle  was used to inject normal saline into the interstitial space outside the thyroid capsule, esophagus, trachea, nerves, and large blood vessels to create an isolation zone of approximately 5 mm in thickness, which made safe margins for major organs to separated the gland to be ablated from the surrounding anatomybefore − Vafter)/Vbefore16.The follow-up included biochemical examination and ultrasonography. The preoperative and each follow-up serum iPTH, calcium, and the volume of hyperplastic parathyroid glands were collected and evaluated. To calculate the volume of the gland, we used the sphere formula: V = π × abc/6, . The volume reduction rate (VRR) was defined by using the formula: VRR =  or Mann–Whitney U test (for skewed data) to compare continuous variables, and the chi-square or Fisher's exact test to analyze categorical variables. All statistical tests were two-sided, and P < 0.05 was considered statistically significant unless stated otherwise.Using SPSS version 26.0 for all statistical analyses. Continuous variables were expressed as mean ± SD for normal distribution or medians (interquartile range) for skewed distribution. Categorical variables were expressed as frequencies. In order to compare the baseline and follow-up data for each patient, paired t-test  or paired-sample Wilcoxon-signed-rank-sum-tests (for skewed distribution) were used for continuous variables. Between the PHPT and the SPHPT groups, using the The study involving human participants was reviewed and approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants and their legal guardians. Research involving human research participants have been performed in accordance with the Declaration of Helsinki.The participants provided their written informed consent to participate in this study.P < 0.001). The level of serum iPTH in SHPT group was 1738.00 (729.45–2637.00) pg/mL, which was much higher than that in PHPT group of 156.30 (120.38–222.25) pg/mL (P < 0.001). In contrast, the two groups did not have notable differences in the preoperative serum calcium and hyperplastic parathyroid volume . For clinical symptoms, the rate of presenting bone pain  and skin pruritus  in SHPT group were significantly greater . Additionally, the two groups did not significantly vary from one another in terms of complications, comorbidities, and the maximum diameter of hyperplastic parathyroid glands.The 57 HPT patients were divided into the PHPT group (n = 48) and the SHPT group (n = 9). This study included a total of 67 hyperplastic parathyroid glands. More patients in the SHPT group had multiple hyperplastic parathyroid glands than in the PHPT group (P < 0.05), compared with the pre-RFA serum iPTH and calcium. The decrease rates of the serum iPTH and calcium in the two groups at 1 month, 3 months, 6 months, and 12 months after RFA were more than 60% and 10% . As for the SHPT group, the serum iPTH of patients at 2 h, 1 day, 1 month, 3 months, 6 months, and 12 months after RFA was 1417.39 ± 1036.84 pg/mL, 1176.68 ± 952.58 pg/mL, 1148.82 ± 1373.18 pg/mL, 990.08 ± 1446.13 pg/mL, 540.63 ± 832.00 pg/mL, 356.53 ± 496.98 pg/mL, respectively compared with the pre-RFA serum iPTH of 1721.91 ± 1083.91 pg/mL, there were significant decreases .For the serum iPTH, in the PHPT group, the serum iPTH of patients at 2 h, 1 day, 1 month, 3 months, 6 months, and 12 months after RFA was 75.37 (31.48–106.68) pg/mL, 37.08 (15.57–64.03) pg/mL, 47.55 (28.25–75.50) pg/mL, 51.05 (25.93–60.60) pg/mL, 43.65 (26.63–62.93) pg/mL, 37.90 (25.93–54.93) pg/mL. Respectively, all of those showed significant decreases compared with the serum iPTH value of 156.30 (120.38 -222.25) pg/mL before RFA . For the serum calcium of SHPT patients at 2 h, 1 day, 1 month, 3 months, 6 months, and 12 months after RFA, it was 2.63 (2.34–2.72) mmol/L, 2.39 (2.10–2.60) mmol/L, 2.36 (2.21–2.54) mmol/L, 2.31 (2.22–2.52) mmol/L, 2.33 (2.22–2.64) mmol/L and 2.34 (2.24–2.73) mmol/L, respectively, all of which showed significant decreases compared with the serum calcium value of 2.73 (2.47–3.25) mmol/L before RFA .The serum calcium of PHPT patients at 2 h, 1 day, 1 month, 3 months, 6 months, and 12 months after RFA was 2.52 (2.36–2.71) mmol/L, 2.41 (2.28–2.59) mmol/L, 2.38 (2.29–2.46) mmol/L, 2.43 (2.32–2.52) mmol/L, 2.39 (2.30–2.48) mmol/L and 2.39 (2.31–2.49) mmol/L, respectively compared with the serum calcium before RFA which was 2.72 (2.56–2.90) mmol/L, there were considerable reductions (P < 0.001). What’s more, the VRR at 3 months, 6 months, and 12 months after RFA were much higher than the VRR at 1 month after RFA (P < 0.001)  mm3, 29.32 (3.61–96.76) mm3. For SHPT patients at 3 months, 6 months, and 12 months after RFA, it was 604.73 (228.76–885.93) mm3, 219.91 (74.81–373.98) mm3, 27.38 ( 8.52–147.26) mm3, which were all significantly smaller than the volume before RFA of 933.05 (395.84–1954.07) mm3 and 1046.15 (490.09–1552.21) mm3 . However, the volume of the two groups at 1 month after RFA was 989.60 (418.88–1759.10) mm3 and 889.84 (404.01–1439.46) mm3, which were not significantly smaller than the preoperative volume .The volume of the ablation area of PHPT patients at 3 months, 6 months, and 12 months after RFA was 529.18 (239.81–997.85) mmP < 0.001, P < 0.001, P < 0.001, P < 0.001, P < 0.001, and P < 0.001).For the VRR at 3 months, 6 months, and 12 months after RFA, it was 44.22 (35.12–50.65)%, 80.45 (73.02–85.23)%, 97.92 (95.65–98.97)% in the PHPT group, and 41.70 ± 14.32%, 75.33 ± 17.25%, 90.83 ± 14.58% for SHPT patients. All of them were significantly higher than the VRR at 1 month after RFA of 3.81 (− 14.57–13.58)% and 2.37 ± 19.58% (P < 0.001)  pg/mL, 1713.00 (401.65–2332.50) pg/mL, 1436.00 (259.15–2108.00) pg/mL, 543.80 (205.90–2368.00) pg/mL, 217.00 (147.20–2017.50) pg/mL, 155.00 (64.55–782.50) pg/mL, 146.00 (43.00–540.50) pg/mL. All of these were substantially higher than those of the PHPT group, which were 156.30 (120.38–222.25) pg/mL, 75.37 (31.48–106.68) pg/mL, 37.08 (15.57–64.03) pg/mL, 47.55 (28.25–75.50) pg/mL, 51.05 (25.93–60.60) pg/mL, 43.65 (26.63–62.93) pg/mL, and 37.90 (25.93–54.93) pg/mL . Regarding the anticipated serum calcium level, the PHPT group had 46 patients (95.8%) and the SHPT group had 7 patients (77.8%), who achieved the clinical success at 6 months after RFA, with no considerable difference (P = 0.113). While the number of SHPT patients dropped to 6 patients (66.7%) at 12 months after RFA, less than the 46 patients (95.8%) in the PHPT group (P = 0.024).For the serologic parameters, 42 cases (87.5%) in the PHPT group achieved the clinical success in the serum iPTH at 6 and 12 months after RFA, which were significantly more than 2 cases (22.2%) and 3 cases (33.3%) in the SHPT group   in the SHPT group that met the clinical success. This was not discernibly different from the 34 cases (70.8%) and 42 cases (87.5%) in the PHPT group (P > 0.05).In the PHPT group, 2 patients (100%) with preoperative skin itching and 7 patients (100%) with bone pain improved after RFA, and the symptoms continued to be relieved until the end of follow-up. The remaining 3 patients who suffered from bone pain also showed relief of symptoms gradually over the follow-up period. Moreover, 6 patients (85.7%) who had neuropsychological symptoms and 8 patients (88.9%) with gastrointestinal symptoms improved after RFA. In the SHPT group, 2 cases (100%) with depression, 6 cases (85.7%) with bone pain, 3 cases (75.0%) with skin pruritus, and 3 cases (75.0%) with anorexia had respectively showed significant improvement after RFA. Clinical symptom relief of RFA was not only satisfying but also not substantially different between the two groups (P = 0.024). After giving regular oral methylcobalamin, 1 PHPT patient and 3 SHPT patients recovered within 3 months after RFA, and the other PHPT patient had relief at the end of follow-up by giving extra dexamethasone nebulized inhalation. As for hematoma, only one SHPT patient (11.1%) developed limitedly within 6 h after RFA, which was controlled by temporary pressure with an ice salt bag. The hematoma was completely absorbed at 1 month postoperatively. The two groups respectively had 1 patient who complained of pain during the RFA, and they were relieved after stopping the RFA and having a short rest. 2 PHPT patients (4.2%) and 2 SHPT patients (22.2%) had transient hypocalcemia after RFA. After the administration of oral calcium, the symptoms were resolved within 1 month after RFA. Furthermore, 13 PHPT patients (27.1%) experienced postoperative transient hypoparathyroidism, and they all returned to normal within 3 months without any additional care. The two groups merely had notable differences in the hoarseness of the mentioned adverse reactions.There were no serious adverse reactions such as dyspnea in the two groups after RFA. Hoarseness suggested that there might be the RLN injury. In the SHPT group, 3 patients (33.3%) developed hoarseness, which was more than the 2 patients (4.2%) in the PHPT group  before and after RFA in PHPT and SHPT patients but also analyze whether there were any significant differences in the treatment outcomes between the two groups. In our research, we found that RFA could substantially decrease the serum iPTH and calcium levels of patients in the two groups. The volumes of hyperplastic parathyroid glands reduced substantially in both groups, and the median VRR in both groups exceeded 95% at the end of the follow-up period. In addition, 87.5% of PHPT patients and 72.2% of SHPT patients achieved the clinical success in VRR at 12 months after RFA. Previous studies by Ralph P. Tufano et al. also showed that RFA was effective in reducing biochemical parameters  and the volume of hyperplastic parathyroid glands to complete disappearance in the PHPT and the SHPT patients, respectively28. That was consistent with the results we obtained indicating that RFA was effective in the treatment of PHPT and SHPT.In recent years, RFA has become a popular alternative to parathyroidectomy for the treatment of PHPT and SHPT patients with apparent symptoms or at risk of affecting other organs, long-term survival, and quality of life due to disease progression. RFA offers the advantages of less trauma, quicker recovery, and lower risk compared to traditional surgical methods35. This research also discovered that the serum iPTH of several SHPT patients increased instead of decreasing after RFA.Intriguingly, although this study demonstrated that RFA was effective in reducing the serum iPTH values of the SHPT patients. Different from the other three indices, the perioperative, postoperative, and reduced serum iPTH levels of SHPT group were considerably higher than those of PHPT group. What’s more, the percentage of SHPT patients who met the normal serum iPTH (15–65 pg/mL) was also much lower than that of the PHPT group. Similarly, when Li, D et al. studied the minimally invasive treatments of the SHPT patients and the PHPT patients with ethanol ablation, microwave ablation, etc., they found that throughout the brief follow-up time, the serum iPTH of the SHPT patients was greater than that of the PHPT patients and normal levels. But also significantly lower than that of the SHPT patients treated with drug-only intervention. The serum iPTH of the SHPT patients would decrease further after long-term follow-up or secondary ablation32. 2. Due to the long-term damage of the kidney and calcium-phosphorus metabolism disorders, multiple parathyroid glands are often stimulated and proliferated in the SHPT patients. While the PHPT patients generally have only one hyperplastic parathyroid gland comparatively37. The SHPT group in our research had significantly more patients with multiple hyperplastic parathyroid glands than the PHPT group. In conclusion, the SHPT patients had more residual parathyroid cells postoperatively. Besides, the poorer body regulation abilities of SHPT patients will cause the disturbance of calcium-phosphorus metabolism to not be completely corrected and continue to stimulate the residual parathyroid cells to increase their proliferation after RFA. We believe that the above-mentioned reasons may result in the serum iPTH of the SHPT group remaining at a higher level than the PHPT group, and appropriately extend the ablation time, increase the ablation power or multiple ablations can be used to achieve the desired outcome37. 3. The effect of RFA is continuous. As well as the immediate dying of cancer cells, some scholars believe that the peak of the effect of thermal ablation on cancer cells generally occurs 4–5 days after RFA. So it takes time for parathyroid cells to complete inactivation41. Whereas in this study, a follow-up time of 12 months might not be sufficient to observe the return of serum iPTH to normal. Therefore, the follow-up time can be appropriately extended in future studies, making it more conducive to exploring whether the serum iPTH will return to the normal level and the long-term efficacy of RFA in the SHPT patients. As for the iPTH rebound phenomenon, some studies pointed out that the body makes adaptive compensatory changes to restore normal calcium homeostasis after RFA. Such as the reduction of ionized calcium activates calcium-sensing receptors on parathyroid cells, which induces PTH secretion and release, or the body develops peripheral PTH resistance43. For this phenomenon, we require a longer time and more patients to explore and clarify.The reasons for this result may be: 1. First and foremost, RFA is performed under US-guided rather than direct vision which is unlike parathyroidectomy. In addition, the parathyroid glands are relatively deep, to protect the nearby main blood vessels, RLN, and minimize postoperative complications to the greatest extent, the parathyroid glands may not be completely ablated, resulting in a small number of parathyroid cells remaining. So the serum iPTH values of HPT patients do not return to normal quickly11. In terms of the volume of the ablation area, this study found that the index in the two groups observably dropped from 3 months postoperatively and the VRR increased greatly over time. The volume of the ablation area at 1 month after RFA did not decrease significantly compared with that before RFA. Because the scope of RFA was generally extended to the edge of the gland 0.5–1 cm , for the purpose of cutting off the peripheral blood supply and preventing recrudescence of HPT after RFA. The VRR of the PHPT group could reach 97.92 (95.65–98.97) % at the end of follow-up time and was similar to that of the SHPT group at 90.83 ± 14.58%. Xinyang Li’s research indicated that the VRR at 12 months after RFA of the PHPT patients was 86%11. In the studies of Wei, Y et al., the VRR of the HPT patients at 12 months after RFA was 94.6–100%, whereas the ablation area of HPT patients who underwent microwave ablation was completely absorbed at 12 months after RFA37. In our study, the decrease in parathyroid volume was slightly different from these prior studies. Probably due to the different ablation methods, different patient conditions, different surgeons, different ablation times, and different power. In future studies, screening patients more carefully, adding the different ablation methods, and extending follow-up times can be used in the more intuitive comparison for which is more effective.However, the two groups had similarities in the values of the last three metrics from the preoperative time to the end of the follow-up of this study. For serum calcium, 46 PHPT patients (95.8%), much more than 6 SHPT patients (66.7%), had achieved the clinical success at 12 months postoperatively. And two other SHPT patients got normal serum calcium after 15 months. We believe that the immoderate serum calcium value is transient, dynamic, and transsexual. It is caused by the body's long-term disease state, poor regulation ability, and oral calcium. Overall, our study on serum calcium is comparable to the earlier research37. Multiple thermal stimulation to RLN during RFA of the multiple hyperplastic parathyroid glands in the SHPT group might lead to the difference between the two groups. In our study, the incidences were higher than those of thyroid ablation (1.5%) and parathyroidectomy (3.9%)44. But all patients with hoarseness improved within the follow-up period after symptomatic treatment, while the researches about thyroid ablation and parathyroidectomy always have patients with permanent hoarseness. The reasons may be that parathyroid glands have nearer locations to the tracheoesophageal groove where the RLN runs than the thyroid, and the RLN is more sensitive to thermal stimulation than mechanical stimulation. We think that minimizing thermal damage to the RLN through sufficient water separation and shorter RFA time is feasible in the future27. Moreover, the patients who developed hypocalcemia, transient hypoparathyroidism, and hematoma after RFA all recovered to normal on their own or with medication within 3 months after RFA. In a word, RFA for PHPT and SHPT is a safe treatment option without serious and permanent complications and adverse reactions.By the end of the follow-up, patients in the PHPT group and the SHPT groups showed great improvement in bone pain, skin pruritus, and digestive tract symptoms. The major complication of this study was hoarseness, for the incidence of the PHPT group was 4.2%, lower than the 33.3% of the SHPT group. The incidences were close to the early published rates of 6–26.671% for the HPT patients treated with ablationThere were several limitations in this study. First, this study had a limited sample size and a brief period of follow-up, which led to a certain selection bias. This type of study needs larger samples, longer follow-up time, and more relevant indicators to fully assess the long-term efficacy of RFA. Secondly, this study only involved the efficacy of RFA on PHPT and SHPT, lacking the comparison of other surgical methods. After that, we can incorporate microwave ablation, laser ablation, parathyroidectomy, and other surgical methods to have a more complete understanding of the efficacy and security of RFA. Thirdly, the chief surgeons in this study included ultrasonographers and endocrinologists in addition to specialists. The varying abilities and experience of doctors may cause bias in treatment, which can be reduced by including only specialist cases.In summary, this study provides evidence that US-guided RFA is a secure and efficient technique to treat patients with PHPT and SHPT. SHPT patients have a more complex preoperative profile. There were better outcomes in terms of iPTH and a lower incidence of postoperative hoarseness for PHPT patients. PHPT and SHPT patients have no considerable difference in terms of serum calcium, the volume of the ablation area, and the VRR. For patients with PHPT or SHPT who are not suitable or refuse parathyroidectomy, RFA may be an alternative.Supplementary Information.