Potential Role of Intravenous Royal Jelly in Reducing Post-hepatectomy Liver Dysfunction: A Randomized Controlled Animal StudY

Preparation of animal models

The experimental animals used in this study were healthy, 10-12 week-old male Wistar rats, weighing 150-200 grams. Sick appeared animals were excluded. During the study, rats were housed individually and provided free access to food and water.

Preparation of intravenous Royal Jelly

Royal Jelly was prepared as described in a previous study.¹⁰ Briefly, Raw Royal Jelly was obtained from local beekeepers (Tawon Gung, Malang, Indonesia) and stored in a freezer at -18°C until preparation of the water-soluble Royal Jelly. One gram of Raw Royal Jelly was homogenized in 10 mL of Phosphate Buffered Saline (Gibco^®, 70011-044) containing 1 M Ammonium Sulfate (Nacalai Tesque, 02619-15). The solution was then centrifuged at 15,000 g for 30 minutes at 4°C. The supernatant was collected and passed through a 0.22-micron syringe filter. The water-soluble Royal Jelly solution was stored in a refrigerator at 4°C until use.

Analysis of Royal Jelly composition

The total sugar content, fatty acid content, total protein content, and amino acid composition of water-soluble Royal Jelly preparations were analyzed using various methods.

Total sugar content was analyzed using the High-Performance Liquid Chromatography (HPLC) method. First, a series of standard sugar mixtures consisting of glucose (Sigma-Aldrich, G7528), fructose (Sigma-Aldrich, F0127), sucrose (Sigma-Aldrich, A2186), lactose (Sigma-Aldrich, 61339), and maltose (Sigma-Aldrich, 63418) were prepared in a 10 mL volumetric flask. Then, 2 mL of water soluble Royal Jelly was added into a 25 mL volumetric flask. Distilled water was added up to half the flask’s volume. Subsequently, 1 mL each of Carrez solution I and II (Chemtest) was added, and the mixture was shaken. The solution underwent sonication for 15 minutes. Distilled water was added up to the calibration mark, followed by homogenization. The solution was filtered using a 0.45-micron syringe filter into a 2 mL vial and injected into the HPLC Refractive Index Detector system (Waters 2414).

Saturated and unsaturated fatty acid content was analyzed using the Gas Chromatography (GC) method. Water-soluble Royal Jelly equivalent to 50 mg of fat was weighed and placed into a 20 mL screw-top vial. MTBE (Merck, 101849) and transesterification solutions were added, and the vial was vortexed for 10 seconds. Afterward, the vial cap was removed, and hexane was added, followed by the neutralizing solution. The mixture was centrifuged at 1750 rpm for 5 minutes. The organic phase was collected into a 2 mL vial and injected into the GC Flame Ionization Detector system (PerkinElmer Claurus 690).

Total protein content was determined using the Kjeldahl method. A total of 0.51 g of water-soluble Royal Jelly was placed in a 100 ml Kjeldahl flask. Then, 2 g of Kjeldahl tablet (Supelco, 1.10958.0250) and 25 ml of concentrated H₂SO₄ (Supelco, 1.00731.2500) were added. The mixture was heated to boiling for 2 hours. After cooling, the mixture was diluted and transferred to a 100 ml volumetric flask. Subsequently, 5 ml of the solution was placed into a distillation apparatus, and 5 ml of 30% NaOH (Supelco, 1.06498.5000) was added. The distillation process was carried out for 10 minutes, using 10 ml of 2% boric acid solution (Supelco, 1.00165.1000) mixed with an indicator as the receiving solution. The condenser tip was rinsed with distilled water. A 0.01 N HCl solution (JT. Baker, 9535-69) was used as the titrant.

A general amino acid analysis was conducted utilizing the Ultra-High Performance Liquid Chromatography (UHPLC) technique. A standard amino acid solution (TCI, A0280), incorporating an internal reference compound, was prepared at a fixed concentration. Subsequently, 1 mL of the water-soluble Royal Jelly solution was transferred into a 20 mL headspace vial and subjected to hydrolysis using hydrochloric acid. The resulting hydrolysate was then poured into a 50 mL volumetric flask, diluted with distilled water to reach the calibration line, and thoroughly mixed. The prepared solution underwent filtration through a 0.2-micron syringe filter, and the filtrate was collected. A second internal standard was introduced before derivatization, after which the solution was injected into the UHPLC Photodiode Array system (Waters Acquity H-Class).

The amino acids cystine and methionine were analyzed using the Liquid Chromatography-Mass Spectrometry (LC-MS/MS) method. A standard series (TRC, C994500 and M260515) was prepared in 2 mL vials using filtered solvents. A total of 0.5 g of water-soluble Royal Jelly was placed into a 20 mL headspace vial, then stored in a cooling water bath before being frozen for 15 minutes. An oxidizing solution was added, followed by homogenization. The preparation was returned to the freezer. Subsequently, sodium bisulfite (Sigma-Aldrich, 243973) was added, and the mixture was left to stand for 3 hours. A hydrolyzing solution was then added, and the mixture was heated in an oven. After cooling, the preparation was diluted with distilled water. The pH was adjusted to 2.2 in a cooling water bath, and homogenization was carried out with distilled water. The solution was centrifuged and filtered using a 0.2-micron membrane filter before injection into the LC-MS/MS Mass Spectrometer system (Sciex Triple Quad 4500).

Tryptophan was analyzed using High-Performance Liquid Chromatography (HPLC). After the preparation of the standard series (TRC, T947210), 0.1 g of water-soluble Royal Jelly was added to a 20 mL headspace vial. A 4.2 M NaOH solution (Merck, 106498) was then added, and the mixture was hydrolyzed at 110°C for 20 hours. The solution was transferred to a 50 mL beaker, and citrate buffer (Merck, 100244) was added. The pH of the solution was adjusted to 4.25, followed by homogenization with distilled water. The solution was centrifuged, and the supernatant was filtered using a 0.45-micron syringe filter before injection into the HPLC Photodiode Array system (Waters Alliance e2695).

Experimental design

In this study, we conducted two phases: a feasibility study and a main study. A feasibility study was performed to determine the extent of liver resection that could be performed while keeping the rats alive. Based on the findings of the feasibility study, the main study was conducted.

In the feasibility study, 10 rats were divided into four groups. The first group of rats underwent laparotomy without liver resection (n=2). The second, third, and fourth groups comprised 70% (n=2), 80% (n=2), and 90% (n=4) of the rats, respectively. The methods for anesthesia, prophylactic antibiotic administration, and pain management were consistent with those described in the main study section. All rats were monitored for survival up to 48 hours post-surgery. If they survived, they were sacrificed after 48 h, and bilirubin and prothrombin times were measured.

For the main study, surgical procedures were performed in the operating room of Airlangga University Animal Hospital. Thirty-two rats were weighed before the liver resection. Anesthesia was induced using 5% inhaled sevoflurane (Sevoflurane, Yarindo Farmatama) and maintained at 3.5% throughout the procedure. Sixteen rats were randomly assigned to receive an intravenous injection of Royal Jelly at a dose of 70 mg/kg via the tail vein immediately before liver resection, with a second dose administered 24 h post-surgery (RJ group). Another 16 rats were randomly assigned to receive an intravenous injection of 0.1 ml phosphate-buffered saline (PBS group) via the tail vein at the same time intervals. The injection via the tail vein was chosen as it is one of the recommended methods.¹¹ Royal Jelly and phosphate-buffered saline were administered in a blinded manner. Prior to surgery, all rats received the prophylactic antibiotic Cefazolin (Cefazolin, Mahakam Betafarma) at a dose of 100 mg/kg. All rats were administered Flunixin Meglumine (Fortis^®, Dong Bang) intramuscularly at a dose of 2.5 mg/kg. After surgery, the rats were housed individually with access to standard food and 10% glucose water. All rats were maintained on a 12-hour light-dark cycle. Flunixin injections were repeated every 12 h, using the same dose and route of administration. Animal suffering was minimized through the administration of analgesics, and the experimental procedures were conducted by experienced veterinary hospital staff. All the rats were sacrificed 48 h after surgery. The euthanasia method employed involves administering an intramuscular injection of a mixture of Ketamine (Ket-A-100^®, AgrovetMarket) and Xylazine (Xyla^®, Interchemie) at an excessive dose (150–15 mg/kg). Subsequently, the previously made laparotomy incision is reopened and extended towards the thoracic cavity. Intracardiac blood samples were collected for bilirubin and prothrombin time analysis. The remaining liver tissue was harvested and fixed in 10% formalin for pathological examination. We report this experiment in accordance with the ARRIVE guidelines.¹² This study has received approval from the Animal Care and Use Committee of the Faculty of Veterinary Medicine, Airlangga University, under license number “2. KEH.047.04.2024” on April 4, 2024.

Surgical technique

Once anesthetized, the abdominal fur of the rats was shaved. Antisepsis was achieved by applying 10% povidone-iodine to the abdominal skin. Surgery was performed in a sterile environment. A 3.5 cm midline incision was made on the cranial abdomen, starting from the xiphoid process and extending caudally. Abdominal wall was retracted to provide visibility of the liver. The ligaments were detached, and resection was performed on the median lobe, left lateral lobe, and caudate lobe following ligation by piercing with 4-0 Silk sutures placed 3 mm from the vena cava. The superior and inferior right lobes were preserved. The surgical area was then irrigated with 3 ml of saline solution. The abdominal wall was closed using continuous 4-0 Polyglactin sutures, and the skin was sutured using interrupted 3-0 Silk ( Figure 1).

Figure 1. (A) Abdominal incision revealing the liver of the rat. (B) The resected liver specimen. (C) Diagram of the rat liver anatomy.

RM: Right Median Lobe; LM: Left Median Lobe; LL: Left Lateral Lobe; AC: Anterior Caudatus Lobe; PC: Posterior Caudatus Lobe; RS: Right Superior Lobe; RI: Right Inferior Lobe. The shaded area indicates the portion of the liver that was resected in this study.

Ki-67 Examination

Forty-eight hours post-surgery, the surviving rats were sacrificed and the superior right lobe of the liver was harvested and fixed in 10% neutral buffered formalin. Paraffin blocks were then prepared from the samples, sectioned at a thickness of 3-5 microns, and mounted onto special slides. Deparaffinization was performed using xylol, followed by rehydration in 96, 90, and 80% alcohol for 2 minutes each. The slides were rinsed with running and distilled water for 5 minutes. Next, the slides were placed in 3% H₂O₂ (Biogear, BGPB-0500) in methanol for 15 minutes at room temperature, rinsed with distilled water for 5 minutes, and heated in Target Retrieval Solution/Citrate Buffer (Biocare Medical, BRRR2004CMX) at pH 6 in a decloaking chamber for 20 minutes at 95°C. The slides were then washed with phosphate-buffered saline (Biogear, BGPBS-002) for 5 minutes. A background snipper was applied for 15 minutes, followed by application of the primary antibody. The study used rabbit monoclonal antibody, clone SP6, and isotype IgG against Ki67 (Biocare Medical, 901-325-060223) at a dilution of 1:50. The expression of Ki67 in hepatocytes was evaluated by identifying positively stained nuclei using an anti-Ki67 antibody. The Ki67 score was based on the percentage of positively stained cells (%) in hotspot areas, analyzed under a binocular light microscope (Olympus CX31) at 400x magnification, and documented using the Olympus DP2-BSW software (see Software Availability statement). Ki67 examination was conducted by an Anatomical Pathology specialist who was blinded to the treatments applied to the specimens under review.

Statistical analysis

The Mann-Whitney test was used to analyze bilirubin and prothrombin time values, while a t-test was used to analyze the Ki67 percentage. Statistical p-value of <0.05 was considered statistically significant. Statistical analysis was performed using SPSS software (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY), and JASP software (JASP Team (2024) JASP (Version 0.18.3) [computer software]) (see Software Availability statement).

Analysis of Royal Jelly components

The water-soluble Royal Jelly used for intravenous preparations contained 1.04% total sugar. Several fatty acids were analyzed but not detected, including dodecanoic acid, decanoic acid, tetradecanoic acid, pentadecanoic acid, heptadecanoic acid, (Z,Z)-9,12-octadecadienoic acid, (Z,Z,Z)-9,12,15-octadecatrienoic acid, eicosanoic acid, docosahexaenoic acid, and tetracosanoic acid. The analysis detected the presence of saturated and unsaturated fatty acids, each at a concentration of 0.01%. Hexadecanoic acid and (9Z)-Octadec-9-enoic acid were detected at levels below 0.01%, while Omega-9 was detected at a concentration of 7.8 mg/100 g. The total protein content was 14.3%. Several amino acids were detected at varying levels. The most abundant amino acids were aspartic acid and glutamic acid. The least abundant amino acids were alanine, cystine, and methionine. The summary of the Royal Jelly component analysis are presented in Table 1. The chromatograms of the conducted analyses are presented in Figures 2-4.

Figure 2. Sugar component chromatogram.

Figure 3. Fatty acid component chromatogram.

Figure 4. Chromatogram of general amino acid component (A); Cystine (B); Methionine (C); and Tryptophan (D).

Feasibility study

All rats that underwent 90% liver resection (4/4) died before the 48-hour mark, with the longest survival time in this group being 17 h. The other three groups survived for 48 hours post-surgery. From the feasibility study, we determined that 70% and 80% of liver resections were feasible. Liver dysfunction was more pronounced in rats that underwent 80% liver resection than in those that underwent 70% resection. Bilirubin and prothrombin time values are listed in Table 2. Based on the results of the feasibility study, we decided to perform 80% liver resection in the main study.

Rat characteristics and survival

The rat body weight, surgery duration, and weight of the resected liver are presented in Figure 5. These parameters showed no significant differences (p>0.05). Four of the 4 rats out of 32 (two from the RJ group and two from the PBS group) died before the scheduled sacrifice time. Therefore, 28 rats were included in this analysis.

Figure 5. Characteristics of the experimental animals.

Body weight (A), surgery duration (B), and resected liver weight (C).

Total bilirubin levels, prothrombin time, and Ki67

In both groups, some rats exhibited elevated total bilirubin levels and prothrombin time compared with rats without liver resection in the feasibility study ( Figure 6). The median total bilirubin level in the RJ group was significantly lower than that in the PBS group (0.4 [0.2-1.5] mg/dl versus 0.9 [0.6-1.9] mg/dl respectively, p<0.001). The median prothrombin time in the RJ group was lower than that in the PBS group (10.35 [9.7-15.2] sec versus 14.5 [9.8-23.1] respectively, p<0.05). In addition, the mean percentage of Ki67 in the RJ group was lower than that in the PBS group (57.14 ± 12.67% versus 71.07 ± 15.95% respectively, p<0.05). Several documented images of the Ki67 examination are shown in Figure 7.

Figure 6. Liver function assessment in both groups: Total bilirubin levels (A), Prothrombin Time (B), and hepatocyte proliferation analysis (C).

Figure 7. Images of Ki67 examination.

The image above illustrates the microscopic structure of liver tissue (400x magnification), showing the distribution of hepatocyte cells examined using Ki67 antibody staining. Hepatocyte cells are considered positive for Ki67 when their nuclei are stained with a brownish color. The staining is observed at varying intensities: weak intensity with light brown staining, moderate intensity with brown staining, and strong intensity with dark brown staining. In contrast, cells that are unstained or negative will have nuclei that remain blue. Image A-D show percentage of positively stained hepatocye with varying intensities. A: 35%; B: 50%; C: 80%, and D: 90%.

Ethical considerations

This study has received approval from the Animal Care and Use Committee of the Faculty of Veterinary Medicine, Airlangga University, under license number “2. KEH.047.04.2024” on April 4, 2024.