Effects of frankincense on experimentally induced renal stones in rats

Abstract Objectives Frankincense (Luban) is a resin obtained from trees of genus Boswellia. The south of Oman hosts Boswellia sacra trees known to have many social, religious and medicinal uses. The anti‐inflammatory and therapeutic potential of Luban has recently attracted the interest of the scientific community. The aim is to study the efficacy of Luban water extract and its essential oils on experimentally induced renal stones in rats. Materials and Methods A rat model of urolithiasis induced by trans‐4‐hydroxy‐L‐proline (HLP) was used. Wistar Kyoto rats (27 males, 27 females) were randomly distributed into nine equal groups. Treatment groups were given Uralyt‐U (standard) or Luban (50, 100 and 150 mg/kg/day), starting Day 15 from HLP induction for a duration of 14 days. The prevention groups were given Luban in similar doses, starting Day 1 of HLP induction for 28 days. Several plasma biochemical and histological parameters were recorded. Data were analysed with GraphPad Software. Comparisons were performed by one‐way analysis of variance (ANOVA) and the Bonferroni test. Results The lithogenic effects of HLP, such as an increase in urine oxalate and cystine, an increase in plasma uric acid and an increase in kidney levels of calcium and oxalate, have all been best significantly reversed by the Luban dose of 150 mg/kg/day. The histological changes of HLP on the kidney tissue including calcium oxalate crystal formation, cystic dilatation, high degree of tubular necrosis, inflammatory changes, atrophy and fibrosis have also been ameliorated by Luban dose of 150 mg/kg/day. Conclusion Luban has shown a significant improvement in the treatment and prevention of experimentally induced renal stones, particularly at a dose of 150 mg/kg/day. Further studies on the effect of Luban in other animal models and humans with urolithiasis are warranted.


| INTRODUCTION
Frankincense or olibanum is the oleogum aromatic resin that is harvested from trees belonging to the genus Boswellia. The major sources of commercial frankincense are B. serrata (India), B. sacra (Oman) and B. carteri (Somalia). The gum resin of the Boswellia trees is obtained by the incision of the stem or branches. The word frankincense is derived from the ancient French name 'frankincense' meaning 'pure incense' and is known in Arabic as 'Luban'.
It has been used as incense since ancient times and in recent years in the preparation of cosmetics and perfumes.
Pharmacologically, it has been shown to possess anti-inflammatory, sedative, anti-hyperlipidaemic, anti-bacterial and anti-cancer properties. [1][2][3] Animal studies and pilot clinical trials support the potential of B. serrata gum resin extract (BSE) for the treatment of a variety of inflammatory diseases. 4 The pharmacological actions of BSE have been mainly attributed to boswellic acids, especially 11-ketoβ-boswellic acid (KBA) and acetyl-11-keto-β-boswellic acid (AKBA), which were proposed to act as selective 5-lipoxygenase  inhibitors. 5 The main components are volatile oils (5-15%), pure resin (55-66%), mucus (12-23%) and boswellic acids (30%). 6,7 Recently, it has been shown that oral administration of the aqueous stem bark extract of B. papyrifera has a dose-and time-dependent nephrocurative effects on acetaminophen-induced kidney damage in rats. 8 In addition, Oleo-gum-resin of B. serrata Roxb induced renoprotective action against gentamicin-induced nephrotoxicity in Albino rats. 9 Also, Zingiber officinale Roscoe (Ginger), gum arabic (AG) and Boswellia have been found to be beneficial adjuvant therapy in patients with acute and chronic renal failure to prevent disease progression and delay the need for renal replacement therapy. 10 2 | MATERIALS AND METHODS

| Animals
Adult male and female Wistar Kyoto (WKY) rats (9-10 weeks old, weighing 240 ± 15 g) were housed in a dedicated room in the small animal house facility of Sultan Qaboos University (SQU). The rats were kept at an ambient temperature of 22 ± 2 C, relative humidity of about 60% and with a 12 h light-dark cycle (lights on at 6:00 a.m.).

| Experimental design
After an acclimatization period of 1 week, the animals were divided into nine groups of six animals each (three males, three females), and the experiment of males and females was conducted in two batches.
Male WKY rats (n = 27) and female WKY rats (n = 27) were randomly distributed into nine equal groups (with three rats each) and treated for 28 days as follows (Tables 1 and 2): group 1, control (Con), continued to receive tap water and the same diet without any treatment until the end of the study. 11 Group 2 was given trans-4-hydroxy-Lproline (HLP) mixed with standard feed for 28 days. For this group, HLP (5% w/w ) was homogenously mixed with the powdered standard pellet diet and given ad libitum. This group is the 'lithogenic control' and was denoted as lithogenic (H). 12 The rats were monitored daily till the end of the experiment. They were weighed on a weekly basis during the experimental period. On the final day of treatment (Day 28), the rats were placed individually in metabolic cages for 24 h. The next day, urine voided in the previous 24 h was collected in sterile tubes, and the total volume voided recorded. The pH of urine samples was measured, and the samples were stored at À80 C pending analysis. The feed and water intake and faeces output were also noted. The rats were weighed and anaesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) administered intraperitoneally. The rats were carefully dissected to expose the abdominal cavity, and blood (about 6 mL) was collected from the inferior vena cava. The blood collected was centrifuged at 900g at 4 C for 15 min to separate plasma. These plasma samples were stored at À80 C until analysis. From each rat, the two kidneys were excised, cleaned by blotting on a filter paper and weighed to record the total kidney weight. A small piece of the right kidney was fixed in formol-saline for subsequent histopathological examinations. The rest of the right kidney and the left kidney were quickly wrapped in aluminium foils and rapidly dipped in liquid nitrogen. These were later transferred to a À80 C deep freezer and stored until analysis. A portion of this frozen kidney tissue was used to prepare 10% w/v renal homogenates in phosphate-buffered saline (PBS); the supernatant was analysed for calcium, oxalate and phosphorus. 14

| Biochemical measurements
The pH of urine was measured using pH test strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany). Urine osmolality was measured using a freezing point osmometer (Osmomat 3000, Gonotec GmbH, Berlin, Germany). All of the tests in plasma, urine and renal homogenates were done using standard ELISA or colorimetric assay kits. Biomarkers of renal function (urea and creatinine) and magnesium in plasma and uric acid and phosphorus in both urine and plasma were all measured using an automated machine (Mindray BS-120 Chemistry Analyzer, Shenzhen Mindray Bio-Medical Electronics Co., Shenzhen, China). Cysteine, sodium and potassium were measured using colorimetric assay kits from Elabscience (Houston, TX, USA). Calcium, oxalate, citrate and phosphate were estimated using colorimetric assay kits from BioVision (Milpitas, CA, USA). 15

| Histopathology
The kidneys were excised, sliced and then routinely processed for histopathology analyses. The pathologists were blinded to the treatment type received by rats. We made 4 μm sections from each kidney sample and stained them individually with three stains: haematoxylin and eosin (H&E), picro-Sirius red (PSR) and periodic acid-Schiff (PAS). All the three stains were purchased from Abcam (Cambridge, UK). A semi-quantitative method was used to score renal tubular necrosis on a scale of 0-4 where 0 = normal, no necrosis; 1 < 10%; 2 = 10-25%; 3 = 26-75%; 4 > 75%. 16 Three 40Â fields were assessed from each kidney section of each rat from the nine groups, and the score was calculated according to the mean percentage. Fibrosis index was calculated using PSR staining, and tubular atrophy was assessed using the PAS staining techniques.
Polarized microscope filter was used to show the birefringence properties of calcium oxalate and Sirius red-stained collagen fibres.
Sirius red-stained slides were analysed following the procedure described by Manni et al. 17 The slides were examined using Olympus BX51 microscope attached to Olympus DP70 camera, and images were acquired using the 40Â objective lens. Three random images of the renal cortex were acquired from each kidney section of each rat from the nine groups and stored in TIFF 24-bit RGB colour image format. Exact camera and microscope settings were retained unchanged during the imaging process. Image analysis of the stored images was performed using ImageJ ® image analysis software (http://rsbweb.nih. gov/ij/). Briefly, the images were transformed into grey scale, and the red-stained collagen was isolated using the hue histogram filter available in 'threshold colour' followed by measuring the isolated area as a percentage. The fibrosis index percentage was calculated to assess the tissues' collagen content by calculating the ratio of the mean Sirius red-stained positive area to the whole mean area of each photomicrograph (for each rat).
Germany) were dissolved in distilled water and administered by oral gavage to the rats at a dose of 50 mg/kg/day. The dose of Uralyt-U was chosen in accordance with previous research. 18 During the treatment period, special care was taken to keep the pH of the urine of rats in the optimal range of 6.2-6.8 by continuous monitoring.

| Preparation of frankincense (B. sacra)
In this study, we used the finest Omani Hojari Frankincense, which is the best in the market of the Sultanate of Oman based on expert opinion. 19,20 Frankincense was obtained from the frankincense tree
The serum biochemical analysis showed that uric acid increased significantly (P < 0.0001) in the lithogenic control group 2 (H) compared with the control group 1 (Con). Thereafter, the highest significant (P < 0.001) reduction in uric acid was achieved in group 8, p (H + B-100), and group 9, p (H + B-150) ( Figure 1D). The kidney homogenate biochemical analysis showed that calcium increased significantly (P < 0.05) in the lithogenic control group 2 (H) compared with the control group 1 (Con). Thereafter, the highest significant showed mild dilatation of renal tubules with intact glomeruli (score 1) ( Figure 2H,I). The lesion scores for each group are shown in Table 3.
The distribution of collagen fibres (stained in red) and the non-   showed mild dilatation of renal tubules with intact glomeruli (score 1).
The lesion scores for each group are shown in Table 3. The distribution of collagen fibres (stained in red) and the non-collagen structures (stained in yellow) was demonstrated using PSR stain in all nine Groups. Also, the fibrosis index percentage was calculated (Table 3)