Urological Science

: 2019  |  Volume : 30  |  Issue : 4  |  Page : 151--156

Olea europaea subsp. Cuspidata and Juniperus procera hydroalcoholic leaves' extracts modulate stress hormones in stress-induced cystitis in rats

Anmar M Nassir1, Ibrahim Abdel Aziz Ibrahim2, Mohamed A Afify2, Naser A ElSawy3, Mohammad Tarique Imam4, Mohammad Hassan Shaheen5, Mohammed Adel Basyuni5, Ammar Bader6, Raed A Azhar7, Naiyer Shahzad2,  
1 Department of Surgery, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
2 Department of Pharmacology and Toxicology, Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
3 Department of Anatomy and Embryology, Faculty of Medicine, Zagazig University, Egypt
4 Departments of Clinical Pharmacy, Faculty of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia
5 Department of Medical Graduates (Interns), Faculty of Medicine, Umm Al-Qura University, Makkah, Saudi Arabia
6 Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia
7 Department of Urology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

Correspondence Address:
Naiyer Shahzad
Department of Pharmacology and Toxicology, Faculty of Medicine, Umm Al-Qura University, Makkah
Saudi Arabia


Objective: To study the effect of Saudi medicinal plant in stress-induced cystitis in experimental rats. Materials and Methods: Seventy-two female Sprague Dawley rats (200–250 g) were divided into eight groups of 9 rats each. Group 1 and 2 are controls assigned nonstressed and stressed, respectively. Other six groups received different treatments for 2 weeks. After the 14 days of treatment, each group was exposed to water-immersion restraint stress (WIRS) for 30 min. Blood samples were collected to measure the corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) levels. The rats were sacrificed, and their urinary bladders were isolated immediately for a histological and immunohistochemical examination. Results: Rats exposed to WIRS had lesions in the urinary bladder, with a significant increase in plasma CRH and ACTH levels in comparison to the nonstressed controls. Bladder cut sections in stressed rats showed no gross structural abnormality in smooth muscle and connective tissue ratio. There were noticeable variations in mast cell (MC) infiltration and activity with a loss of more than 20% of cellular staining and a significant increase in the number of red blood cell-filled blood vessels. Our findings showed that supplementation of Olea europaea leaf extract (OEE) or Juniperus procera leaf extract (JPE) reduced the MC infiltration and significantly reduced stress hormones compared to the stressed controls. Conclusions: The present study demonstrated that OEE/JPE alone and their combination have a potential protective effect against stress-induced cystitis in rats. The underlying mechanism of the present study also resulted in a decrease in CRH and ACTH stress hormones.

How to cite this article:
Nassir AM, Aziz Ibrahim IA, Afify MA, ElSawy NA, Imam MT, Shaheen MH, Basyuni MA, Bader A, Azhar RA, Shahzad N. Olea europaea subsp. Cuspidata and Juniperus procera hydroalcoholic leaves' extracts modulate stress hormones in stress-induced cystitis in rats.Urol Sci 2019;30:151-156

How to cite this URL:
Nassir AM, Aziz Ibrahim IA, Afify MA, ElSawy NA, Imam MT, Shaheen MH, Basyuni MA, Bader A, Azhar RA, Shahzad N. Olea europaea subsp. Cuspidata and Juniperus procera hydroalcoholic leaves' extracts modulate stress hormones in stress-induced cystitis in rats. Urol Sci [serial online] 2019 [cited 2020 May 31 ];30:151-156
Available from: http://www.e-urol-sci.com/text.asp?2019/30/4/151/263646

Full Text


Cystitis is an inflammatory bladder disease manifested as pelvic, bladder, and urethral pain. The most common cause of chronic cystitis is interstitial cystitis (IC)/painful bladder syndrome (PBS);[1] in addition, it has significant disruption in voiding effects. IC/PBS affects all age groups of either sex.[1] The International Continence Society describes it as “the complaint of suprapubic pain related to bladder filling, accompanied by other symptoms such as increased day and night-time frequency in the absence of proven urinary infection or other obvious pathology of the lower urinary tract.” The prevalence of IC/PBS is 14-fold higher in females than in males.[2]

To date, the etiology of this disease is not completely understood. Moreover, some studies suggest that individuals with IC/BPS are 4.37 times more likely than controls to have a prior diagnosis of stress or anxiety disorders, and this strong association might identify important clues to the pathogenesis and pathophysiology of IC/BPS.[3] Stress causes a sympathetic effect leading to the initiation of inflammatory processes in IC. Stress has shown to activate bladder mast cells (MCs), apparently through the action of at least some sensory neuropeptides.[4] These outcomes have implications for the pathophysiology and possible treatment of IC.

Stress is known to initiate the hypothalamic–pituitary–adrenal axis [5] and thus can influence defects [5] related to the immune system, especially neuroinflammatory disorders. These impacts are most likely intervened through both psychoneuroimmune and neuroendocrine-resistant associations that add to the aggravation and inflammatory reactions [6] and intensify certain neuroinflammatory conditions, such as headaches, neurogenic pruritus, and IC, all of which have been associated with MC initiation.[6],[7]

Currently, there is a huge and wide interest in using natural medicinal products in the prevention and treatment of many illnesses.[8] Historically, in Saudi traditional medicine, Olea europaea (OE) and Juniperus procera (JP) have been used as a combination for treating inflammatory bladder conditions and prostatic hypertrophy for a long time. The leaves and fruits of OE contain several pharmacological active constituents including polyphenols and flavonoids.[9] The most abundant compound in olive leaf extract (OLE) is oleuropein, followed by hydroxytyrosol. The main polyphenols are luteolin, apigenin, and verbascoside.[10] Other bioactive constituents of OLE include triterpenes, such as oleanolic acid,[11] rutin, and diosmin.[12],[13],[14] Oleuropein is considered to have great potential as an antioxidant and food additive but also as a possible therapeutic option.[8],[9],[10],[11],[12] A wide range of studies on oleuropein have been performed using in vitro assays, animal models of disease, and human volunteers, to explore possible beneficial effects for human health.[15]

JP knew in Arabic (Ar'ar) is used for many medical purposes.[16] It is widely distributed in the southern part of the Arabian peninsula (Saudi Arabia and Yemen), Eastern Africa (including Sudan and Zimbabwe), and to some extent, the Americas.[16],[17] Different parts of the plant (leaves, berries, and bark) are used for the treatment of various diseases such as asthma, sore throat, pneumonia, tuberculosis, bronchitis, hyperglycemia, eczema, and skin rash. It has also used for the treatment of jaundice and in wound healing.[18],[19],[20] In traditional medicine, Juniperus species are used as remedies against the common cold, urinary infections, urticaria, dysentery, hemorrhage, rheumatic arthritis, and to relieve menstrual pain in many parts of the world.[21],[22],[23]

This study aimed to estimate the possible protective effect hydroalcoholic extracts of OE subsp. Cuspidata and JP leaves against stress-induced cystitis in female Sprague Dawley rats and the effect of treatment on the stress hormones, i.e., corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) in rats exposed to water immersion-restraint stress (WIRS).

 Materials and Methods

This study is an interventional study using animal models. The rats were divided randomly into eight groups and keep in cages (5 rats/cage) in the animal house of Umm Al-Qura University, Faculty of Medicine.


Seventy-two adult female Sprague-Dawley rats approximately 4 months of age at study initiation, weighing 200–250 g, were got from the animal house of the faculty of medicine Umm Al-Qura University. The rats were individually housed in high-quality polyethylene cages with stainless steel wire mesh flooring in a controlled environment at 23°C–25°C and 50% humidity with a 12-h artificial light cycle on a 12:12-h dark-light cycle (07.00–19.00 lights on). Food was maintained on a pellet diet and tap water ad libitum during the entire period of the study. At the end of the experimental study, it was sacrificed rats for rapid urinary bladder isolation for histological and immunohistochemical examination.

Plant material collection and extraction

The leaves of OE subsp. Cuspidata and JPwere collected in 2016 at Wadi Thee Ghazal, Taif Region, Saudi Arabia (GPS Coordinates: 21.092098, 40.379891). The voucher specimens were preserved in the Herbarium of Pharmacognosy Lab at Umm Al-Qura University with the number (UQU-Med 2015/1 for OE subsp. Cuspidata and UQU-Med 2015/2 for JP). The extracts were prepared by maceration and stirring of 50 g of each dry and powdered plant material in 1 L of a 50% hydroalcoholic solvent (50% ethanol + 50% distilled water v/v). Then, the extracts were filtered separately and dried using a rotary evaporator. The yield of OEand JPextracts (JPE) was 19.3% and 17.8%, respectively. Furthermore, these extracts were stored in a refrigerator for subsequent experimental use.

Study protocol

A total of 72 healthy Sprague Dawley female rats were divided randomly into 8 groups of 9 rats in each group. Blood samples were collected at day zero of treatment. The treatment was given according to the following treatment plan:

Group 1 – Normal control group treated with physiological saline orally 10 ml/kg body weight not subjected to stressGroup 2 – Treated with physiologic saline orally 10 ml/kg body weight and subjected to stress

Group 3 Administered pentosan polysulfate 30 mg/kg body weightGroup 4 – Administered OEextract (OEE) 500 mg/kg body weightGroup 5 – Administered OEE 250 mg/kg body weightGroup 6 – Administered JPE 500 mg/kg body weightGroup 7 – Administered JPE 250 mg/kg body weightGroup 8 – Combined extracts of JPE and OEE administered each 250 mg/kg body weight.

All the drugs were administered using oral gavage for 14 days and all the rats were subjected to stress except normal control (Group 1). The rats were deprived of food for 24 h before the experiment. Stress was induced by placing the animals into a plexiglass immobilizer (Harvard Instruments, Cambridge) located on a bench top and restrained for 30 min at room temperature. The experiment was done at the same time of the day to avoid the diurnal rhythm. After exposure to stress, the rats were anaesthetized by injecting both ketamine (100 mg/kg) and xylazine (15 mg/kg). The rats were then sacrificed, and blood samples were collected in different vacutainer from the inferior vena cava. CRH and ACTH values were determined by automated ELISA Kits.

Histological technique

Urinary bladder specimens were taken from the pelvis and were cut into sections (~ 4–5 μm thickness). Proper fixation was done using 10% neutral-buffered formaldehyde. The tissues were then dehydrated, cleared in xylol, and embedded in paraffin wax. Finally, the bladder sections were stained with hematoxylin and eosin stain [12] and the 0.5% acidified stain (pH <2.5) toluidine blue (Sigma, St. Louis). Two individuals independently counted all MCs, each blinded to the experimental conditions. Each cross-sectional specimen was examined for (a) total number and activity of MCs: (1) resting (not degranulated), (2) partially activated (released < 20% of granules), and (3) fully activated (released >20% of granules). A total of 2–4 sections were counted for each bladder specimen, and the mean was calculated using image analyzer (Leica Imaging System, LTD) (b) bladder inflammation was measured according to the following score criteria: (1) mild infiltration with or without mild edema, (2) moderate infiltration with moderate edema, and (3) severe infiltration with severe edema.

Statistical analysis

The data were expressed as the mean ± standard error of mean and were analyzed using the Statistical Package for Social Sciences (SPSS for Windows®, version 23.0, IBM Corporation). Statistical comparisons were performed by a two-way analysis of variance. The results were considered statistically significant if P < 0.05.


Corticotropin-releasing hormone

In Group 2 (stress control), the value of CRH was highly elevated, which indicated severe stress and inflammation. Group 4 (OE 500 mg/kg)-treated rats showed a highly significant (P = 0.004) decrease. Group 5 (OE 250 mg/kg; P = 0.0492), Group 6 (JP 500 mg/mg; P = 0.039), and Group 8 (OE + JP 250 mg/kg each; P = 0.048) exhibited a significant decrease in CRH values. There was a nonsignificant numerical decrease in Group 3 (PPS 30 mg/kg; P = 0.069) and Group 7 (JP 250 mg/kg; P = 0.062). As a consequence of these values, the rats that were exposed to WIRS had IC and a significant increase in the plasma CRH levels in comparison to the nonstressed control [Table 1].{Table 1}

Adrenocorticotropic hormone

In Group 2 (stress control), the ACTH value was highly elevated which indicated severe stress and inflammation. Group 4 (OE mg/kg; P = 0.004) and Group 6 (JP 500 mg/kg; P = 0.003) showed a highly significant decrease in ACTH values. Group 5 (P = 0.042), Group 7 (P = 0.039), and Group 8 (P = 0.048) showed significant decrease in plasma ACTH while Group 3 had no significant changes [Table 1].


Normal control (saline-treated) groups showing normal histology of the urinary bladder muscle layer, urothelium, and lamina propria were subdivided into a superficial dense irregular layer and a deep loose layer containing small blood capillaries (average MC number = 1) [Figure 1]a and [Figure 1]b. Urinary bladder sections in stress model rats showed no gross structural abnormality in smooth muscle and connective tissue ratio but obvious variations in MC filtration, with a MC activation loss of more than 20% cellular staining and a significantly greater number of red blood cell-filled vessels. Multiple detachments, vacuolation, and focal decrease in the urothelium (uroepithelium) cell layers and disrupted lamina propria containing dilated blood vessels and leukocytes infiltration with multiple MCs (average MC number = 9 with Grade 3 inflammation) were observed [Figure 2]a and [Figure 2]b. In Group 3, rats showed minimal detachment of urothelium and minimal infiltration without edema (average MC number = 3 less than Grade 1 inflammation) [Figure 3]a and [Figure 3]b. In Group 4, mild infiltrations in lamina propria, detached mucosa with vacuoles, and mild edema (average MC number = 4 with Grade 1 inflammation) were observed [Figure 4]a and [Figure 4]b. Group 5 showed dilated and congested vessels and MCs in urothelium and lamina propria (average MC number = 6 with Grade 2 inflammation) [Figure 5]a and [Figure 5]b. Group 6 showed dilated and slightly congested vessels, MCs in urothelium and lamina propria, and multiple detached and vacuolated (average MC number = 5 with Grade 1 inflammation) [Figure 6]a and [Figure 6]b. Group 7 showed minimal infiltration with normally appearing urothelium and few MCs (average MC number = 2–3 Grade 0.5 inflammation/non-inflammatory) [Figure 7]a and [Figure 7]b. Group 8 rats showed minimal infiltration with multiple detachments of urothelium and few MCs (average MC number = 3 with Grade 1 inflammation) [Figure 8]a and [Figure 8]b.{Figure 1}{Figure 2}{Figure 3}{Figure 4}{Figure 5}{Figure 6}{Figure 7}{Figure 8}


The present study investigated that the possibility of OE/JP extracts could minimize stress-induced cystitis in rats, by pretreatment of the extracts. The current work is the first experimental study planned to estimate whether the supplementation of leaves extract of OEsubsp. Cuspidata, JP, and the combination ofOEsubsp. Cuspidata and JP would have a protective effect against stress-induced cystitis with physiological disorders and histological harms in rats.

Various rodent models of cystitis [4],[5] and a naturally occurring feline IC model [6] showed increased MC counts and activation. These models were associated with inflammation and hyperalgesia in the pelvic region. These studies revealed MC involvement in neurogenic bladder inflammation and pain in cystitis.[7] The present study showed that the administration of extracts OEsubsp. Cuspidata, JP, and the combination ofOEsubsp. Cuspidata and JP for 2 weeks produced a significant decrease of serum ACTH and CRH levels. Histopathologically marked alterations of the urinary bladder structure with a notable decrease in MC number and activation were observed. The present work showed that the treatment of rats with OEsubsp. Cuspidata and JPleaves' extracts and their combination for 2 weeks attenuated the physiological and histopathological changes in the urinary bladder of rats induced by water-immersion stress. This indicated the effectiveness of these extracts in the prevention of stress-induced cystitis. The main ingredient of the olive leaves is oleuropein, one of secoiridoid monoterpenes, which is thought to be responsible for pharmacological effects. Furthermore, the olive leaves contain triterpenes (oleanolic and maslinic acid), flavonoids (e.g., luteolin, apigenin, rutin), and chalcones.[24],[25] Due to its chemical composition, olive leaves are considered one of the most powerful innate antioxidants.

In vitro, oleuropein has high antioxidant activity compared to a hydrosoluble analog of tocopherol,[26] as do other constituents of olive leaves.[27] It was shown that total olive leaves' extracts had antioxidant activity more potent than Vitamin C and Vitamin E, due to the synergy between flavonoids, oleuropeosides, and substituted phenols.[8]

Other studies [15],[16] have evaluated the antioxidant properties of oleuropein on ethanol-induced oxidative damage and its beneficial effects on the liver function of Sprague Dawley male rats. They reported that oleuropein during ethanol treatment in rats resulted in higher antiperoxidative enzyme activity, catalase, and inhibited toxicity to the liver as monitored by the reduction in alanine aminotransferase and aspartate aminotransferase levels and thiobarbituric acid reactive substance concentration. They suggested that oleuropein possesses beneficial antioxidant effects against ethanol-induced liver toxicity.

Al-Attar et al.[14] investigated the influence of olive (OE) leaves extract on thioacetamide (TAA)-induced hepatic cirrhosis in Wistar male rats. They demonstrated that the extract of olive leaves possesses hepatoprotective properties against TAA-induced hepatic cirrhosis by inhibiting the physiological and histopathological alterations. Moreover, they suggested that the hepatoprotective effects of olive extract may be attributed to its antioxidant activity.

The petroleum ether fraction of JP showed significant hepatoprotective activity when investigated against CC14-induced liver injury in Wistar male rats.[28] The hepatoprotective activity was estimated throughout the quantification of biochemical parameters and confirmed by histopathology analysis. Different fractions obtained from the aerial parts of JPshowed significant activity as hepatoprotective when investigated against CC14-induced liver injury in Wistar male rats.[29] The hepatoprotective activity was evaluated through the quantification of biochemical parameters and confirmed using histopathological study.


The most important findings in the current work are the observation that OEsubsp. Cuspidata and JPleaves' extracts and their combination were effective in attenuating stress-induced cystitis that was confirmed by biochemical analysis and histopathological examination. It may, therefore, be suggested from evidence in the current work that the supplementations of the OEsubsp. Cuspidata and JPleaves' extracts may give some beneficial results for IC. Moreover, this study suggests that the supplementation of these extracts may act by modulating stress hormones and could be used as excellent adjuvant support in the treatment of cystitis induced by stress. Finally, physiological, biochemical, and histopathological studies are needed to explore the exact mechanism of these extracts and their natural chemical constituents against other related pathogenic factors.


The authors gratefully acknowledged the college administration for providing the facility to work.

Financial support and sponsorship

This study was financially supported by the ICSR (Institute of Scientific Research and Revival of Islamic Heritage) at Umm Al-Qura University, Makkah, Saudi Arabia (Project ID: 43509010).

Conflicts of interest

There are no conflicts of interest.


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