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Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy
Journal Pre-proofs Original Research Article Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy Mohadeseh Ostovar, Abolfazl Akbari, Mohammad Hossein Anbardar, Aida Iraji, Mohsen Salmanpour, Salar Hafez Ghoran, Mojtaba Heydari, Mesbah Shams PII: DOI: Reference:
S2095-4964(19)30117-7 https://doi.org/10.1016/j.joim.2019.12.002 JOIM 133
To appear in:
Journal of Integrative Medicine
Received Date: Accepted Date:
27 December 2018 20 May 2019
Please cite this article as: M. Ostovar, A. Akbari, M.H. Anbardar, A. Iraji, M. Salmanpour, S.H. Ghoran, M. Heydari, M. Shams, Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy, Journal of Integrative Medicine (2019), doi: https://doi.org/10.1016/j.joim.2019.12.002
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© 2019 Shanghai Changhai Hospital. Published by ELSEVIER B.V.
JIM-12-2018-OA-ER-0398 Original Research Article
Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy Mohadeseh Ostovar1, Abolfazl Akbari2, Mohammad Hossein Anbardar3, Aida Iraji4, Mohsen Salmanpour5, Salar Hafez Ghoran6, Mojtaba Heydari7, Mesbah Shams8
1. Department of Traditional Persian Medicine, School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 2. Department of Physiology, School of Veterinary Medicine, Shiraz University, Shiraz 73, Iran 3. Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 4. Central Research Laboratory, Shiraz University of Medical Sciences, Shiraz 73, Iran 5. Department of Pharmaceutics, Center for Nanotechnology in Drug Delivery, Pharmacy School, Shiraz University of Medical Sciences, Shiraz 73, Iran 6. Department of Chemistry, Golestan University, Gorgan 49, Iran 7. Research Center for Traditional Medicine and History of Medicine, Iran School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 8. Endocrine and Metabolism Research Center, Shiraz University of Medical Sciences, Shiraz 73, Iran
ABSTRACT
Objective: This study investigated the biochemical, histopathological and physiological effects of Citrullus colocynthis on peripheral neuropathy in rats with streptozotocin (STZ)-induced diabetes. Methods: Seventy adult male Sprague-Dawley rats were included in the present study. Diabetes was induced in 60 rats, with a single intraperitoneal injection of STZ (65
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mg/kg). After 4 weeks, the diabetic rats were assessed for neuropathy. Then, the diabetic rats with neuropathy were randomly divided into 6 groups for a 4-week treatment with gabapentin, oral administration of C. colocynthis fruit pulp powder (100 and 300 mg/kg per day), topical preparations as oil-based solution and ointment, or placebo. Changes in metabolic, physiological, biochemical and histological parameters were considered as treatment outcomes. Results: Metabolic outcomes (body weight and blood glucose level) were improved in the C. colocynthis-treated groups as compared to placebo. Tail-flick and hot-plate tests also had lower latency in the C. colocynthis-treated groups. Measurement of oxidative stress markers (malondialdehyde, superoxide dismutase and catalase) showed the antioxidant effect of C. colocynthis. Histological evaluation of the sciatic nerve showed that C. colocynthis decreased the number of demyelinated and degenerated nerve fibers. Among the C. colocynthis-treated groups, the one receiving 100 mg/kg per day oral powder had the best treatment outcomes. Conclusion: The present study showed that C. colocynthis fruit, through its antioxidant and hypoglycemic activities, has a positive effect in the treatment of diabetic neuropathy. Please cite this article as: Ostovar M, Akbari A, Anbardar MH, Iraji A, Salmanpour M, Ghoran SH, Heydari M, Shams M. Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy. J Integr Med. 2019; Epub ahead of print. Received December 27, 2018; accepted May 20, 2019.
Keywords: Citrullus colocynthis; Diabetic neuropathy; Diabetes mellitus; Herbal medicine; Natural medicine; Traditional Persian medicine
Correspondence: Mesbah Shams, MD; E-mail address: [email protected]
1. Introduction
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Diabetes mellitus (DM) is a metabolic disease caused by problems in production and/or use of insulin and is diagnosed by high level of blood glucose. It is one of the most common chronic medical conditions encountered in clinical practice. The global prevalence of DM is known to be rapidly increasing as a result of aging populations and lifestyle changes. The International Diabetes Federation estimated that 415 million adults had diabetes in 2015 and the number would increase to 642 million by 2040 [1]. DM can lead to serious complications affecting multiple organs such as heart, blood vessels, nerves, eyes and kidneys. Diabetic neuropathy (DN), as a chronic microvascular complication of diabetes, results in significant morbidity and mortality [2]. About half of people with diabetes suffer from neuropathy [3]. The most common type of DN is distal symmetrical sensory neuropathy, or known in polyneuropathy. Studies on the etiology of DN have identified several biochemical mechanisms of nerve and neurovascular damage, among which, oxidative stress is thought to be the most important [4]. Both chronic and acute hyperglycemia cause oxidative stress in the peripheral nervous system, leading to an acceleration of DN development [5]. Due to the limited efficacy of the drugs commonly used to treat DN pain, phytotherapy is a common alternative therapy and is a popular treatment (amongst different complementary and alternative medicines) for many chronic diseases and their complications [6–8]. Citrullus colocynthis L. (Cucurbitaceae), also known as bitter apple, a well-known plant of ancient medical science, is a desert plant found in Africa, the Middle East and Asia [9]. In Iran, it grows in the southwest, southeast, central and eastern regions [10]. In traditional Persian medicine (TPM) references, such as Liber Continens written by Rhazes (865–925 A.D.) [11], Canon of Medicine by Avicenna (980–1037 A.D.) [12], and the Storehouse of Medicaments by Aghili Shirazi (1670– 1747 A.D.) [13], C. colocynthis is prescribed for patients with nerve-originating pain [14]. Recent studies have investigated many pharmacological effects of C. colocynthis, including antimicrobial, anti-inflammatory, antioxidant, analgesic, local anesthetic, hypolipidemic and blood glucose-lowering effect [15]. Also, its positive effect on diabetes and diabetic nephropathy, as one of its main complications, has been reported [16]. Moreover, we have recently reported the clinical efficacy of the topical use of C. colocynthis in patients with painful DN, showing its efficacy for decreasing pain and
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improving nerve function [17]. However, the underlying mechanism of its protective effect in DN is not well studied. This study investigates the underlying mechanism of the neuroprotective effect of C. colocynthis (considering the physiologic, biochemical, oxidative stress and pathologic outcomes) on DN in an animal model.
2. Material and methods
2.1. Ethical considerations All experimental protocols were approved by the institutional animal ethics committee of the Shiraz University of Medical Sciences, Shiraz, Iran (approval No. IR.sums.REC.1396.S12). Also, the recommendations of the European Council Directive (86/609/EEC) of November 24, 1986, regarding the standards in the protection of animals used for experimental purposes were followed. 2.2. Plant material Dried C. colocynthis fruits were purchased from a medicinal plant market in Shiraz; they were from a batch that had been collected in the Kazerun area of Southern Iran. Following authenticated by Dr. M. M. Zarshenas (Department of Phytopharmaceuticals, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran), a voucher number was specified for the sample (No. PM776). The sample was preserved in the herbarium of Pharmacy School at Shiraz University of Medical Sciences. 2.3. C. colocynthis formulations In order to evaluate the role of preparation on therapeutic efficacy, three types of C. colocynthis-containing products were prepared. 2.3.1. Powder of fruit pulp The pulp of dried C. colocynthis fruits was ground in a mechanical grinder. The resulting powder was sieved using # 50 mesh and kept in a freezer. 2.3.2. Oil-based solution and ointment The milled, dried C. colocynthis fruits were gently stirred with distilled water for 10 min. The mixture was concentrated using a rotary evaporator and then freeze-dried to obtain a fine dry powder. Then the extract was used to prepare oil-based solution and ointment as follows [18]. 2.3.2.1. Oil-based solution
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The extract of C. colocynthis fruits was boiled in sesame oil at 100 oC for 15 min, as traditionally recommended, followed by evaporation method to remove the water portion [18]. 2.3.2.2. Ointment The extract of C. colocynthis fruits was reconstituted in distilled water and emulsified in lanoline as 5% (w/w) product using soybean lecithin emulsifier. 2.4. Qualitative analysis by thin-layer chromatography Cucurbitacin E (CE), as one of the most abundant chemical compounds isolated from C. colocynthis fruit, was used as the reference compound (Sigma Chemical Co., > 98% purity; St. Louis, Mo, USA) [19]. In order to detect CE in the extract of C. colocynthis fruit, thin-layer chromatography (TLC) was performed on normal phase silica gel G254 plates (Merck, Germany). First, standard concentrations of CE (1 mg/mL) and C. colocynthis extract (10 mg/mL) were prepared. Having carefully examined various solvent systems, including n-hexane:ethylacetate, chloroform:acetone, chloroform:methanol and ethylacetate:methanol, the mixture of ethylacetate:methanol was selected to elute samples on the TLC sheet. Samples on the TLC sheets were detected under an ultraviolet (UV)254 lamp and visualized using anisaldehyde/sulfuric acid reagent followed by heating. All experiments were carried out in duplicate. 2.5. Quantitative analysis of CE using external standard 2.5.1. Reagents and instrumentation Standard CE was purchased from Sigma Aldrich Co. (high-performance liquid chromatography [HPLC] grade; ≥ 98% purity; St. Louis, Mo, USA). Methanol (HPLC grade) was purchased from Merck, Germany. High-purity water was prepared using Millipore purification system. The HPLC (KNAUER®) was equipped with quaternary K-1001 controller pumps and a K-2600 UV absorbance detector. The injector was matched with a 20 µL sample loop. 2.5.2. Reverse-phase HPLC condition The binary mobile phase consisted of 60% water and 40% methanol to reach the high polarity (100% methanol). The stainless steel analytical column used had the following properties [20]: Lichrosphere-100, reverse-phase C-18, 250 mm × 4.6 mm inner diameter, 5 µm, with precolumn. The UV-visible light detector was set at 254 nm as previously discussed [20]. Moreover, the flow rate and pressure were set at 1.0 mL/min
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and 190–220 bar, respectively. Having optimized the chromatographic conditions, all analyses were carried out at the room temperature. The volume of all injections into the column was 10 µL and the detecting took place in triplicate. 2.5.3. Preparation of standard CE solution, calibration curve and linearity About 1 mg of standard CE was precisely weighted and dissolved in 1 mL methanol (HPLC grade) to reach the final concentration of 1000 µg/mL. The calibration curve for CE was prepared using concentrations of 1 to 1000 µg/mL. The linear approximation was calculated for concentration range of 1 to 500 µg/mL. 2.5.4. Preparation of C. colocynthis extract About 1 g of the extract of C. colocynthis was weighed and dissolved in 50 mL methanol (HPLC grade). After sonication for 15 min, a cloudy solution was obtained. This solution was centrifuged and then filtered through Whitman quantitative filter paper (ashless, Grade 44, Merck, Germany) to yield the clear solution. 2.5.5. Estimation of CE from the extract of C. colocynthis The recorded area of all peaks was used for quantification of CE in the plant extract. In order to determine the amount of CE, the area values of each concentration were fitted to a calibration curve. 2.5.6. Precision and accuracy Precision was evaluated at two levels in order to verify the repeatability of retention time: interday and intraday. All experiments for this study were analyzed through five determinations at concentration of 5 µg/mL in one laboratory on one day. On the second day, the same samples were analyzed following the same methods. Outcomes were expressed in terms of standard deviation (SD) and repeatability SD (RSD%). The accuracy of the analytical method was expressed as recovery (%) and was measured by using standard addition method [21]. Three concentrations of standard (1, 50 and 100 µg/mL) were spiked into the same amount of examination samples (the prepared extract of C. colocynthis). 2.6. Antioxidant activity Analysis of the antioxidant activity of C. colocynthis was carried out according to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods, as previously described [22,23]. The antioxidant activity was determined by measurement of the decrease in the absorbance at 517 nm. Several aliquots, containing different sample concentrations, were tested and
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the results were reported by the percentage of residual or inhibited DPPH against the concentration of the sample. A stock solution of 110 μmol/L DPPH was prepared fresh by dissolving the appropriate amount of reagent in methanol. Twenty microliters of different concentrations of the sample extract or standard solution were mixed with 180 µL of a methanolic solution of DPPH and kept in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against methanol as blank. Percentage of DPPH reduction was determined using the following equation: percentage of DPPH reduction = (A0 – A1)/A0) × 100, where A0 is the absorbance of the control, and A1 is the absorbance in the presence of the sample. All determinations were performed in triplicate. 2.7. Determination of total phenolic content by Folin-Ciocalteu assay The total phenolic content of C. colocynthis was determined following the procedure of Waterhouse [24]. Diluted extract or blank liquid was mixed with 3.16 mL of water and 0.2 mL Folin-Ciocalteu reagent for 8 min at 22 °C. Then, 0.6 mL saturated sodium carbonate solution was added and the mixture was shaken vigorously for 20 s. The reaction was incubated in the dark at 22 °C for 2 h before measuring the absorbance at 765 nm using a UV-visible spectrometer (Cary 100 UV-Vis; Agilent Corporation, Santa Clara, CA, USA). A standard curve was prepared based on gallic acid standard solutions ranging from 0 to 1000 mg/L. The concentration of the total phenols was expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/gE). 2.8. Experimental animals Seventy healthy adult male Sprague-Dawley rats (Rattus norvegicus; [220 ± 15] g; aged 7–8 weeks) were colony-bred in the Animal House Center, Shiraz, Iran. SpragueDawley rat is a strain of albino rat used extensively in medical research because of its calmness and ease of handling. During the study period, standard pellet diet and water were given to experimental animals. They were housed (5 rats per cage) in the animal room under constant temperature at (22 ± 2) °C and humidity at (55 ± 5)%, with 12hour light and dark cycle and had free access to laboratory food and tap water. Special care was provided to minimize animal suffering and to reduce the number of animals used to the minimum required for statistical accuracy. Animals were acclimatized to
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laboratory conditions before the tests. All the experimental procedures were carried out between 09:00 a.m. and 09:15 a.m. 2.9. Induction and assessment of diabetes and neuropathy The diabetes was induced by a single intraperitoneal injection of a prepared solution of streptozotocin (STZ; 65 mg/kg body weight, Sigma-Aldrich, Germany) in 0.1 mol/L sodium citrate buffer (pH = 4.5); the control rats were injected with citrate buffer alone [25]. To prevent the drug-induced hypoglycemia, a solution of 5% glucose overnight was given to rats. To confirm diabetes, at the third day after STZ injection, the blood samples were collected via the rat tail vein technique using heparinized capillary glass tubes, and plasma glucose levels were measured by the enzymatic GOD-POD diagnostic kit method (Accurex®, India) [26]. The animals were considered diabetic if their blood glucose values were above 300 mg/dL. Then, after 4 weeks, neuropathy was confirmed using hot-plate test in the diabetic rats, by the Eddy and Leimbach method [27]. The animals were included in the study if the latency was more than 5 s; this latency indicates sensory dysfunction in rats. Fig. 1 shows the diagram of the experimental design.
Fig. 1. The diagram of the experimental design.
2.10. Experimental design and protocol To evaluate the effects of C. colocynthis in DN, the animals confirmed to have diabetes and neuropathy were divided into seven groups so that each group included ten rats as follows. Group 1: control rats or vehicle group, received a 0.1 mol/L citrate buffer (pH = 4.5) injection only at the start of the study and then distilled water orally by gavage
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for 4 weeks; Group 2: diabetic rats, as a diabetic control group, that received distilled water (0.05 mL twice a day) for 4 weeks orally by gavage; Group 3: diabetic rats, serving as a positive control, that received gabapentin daily by oral gavage, for 4 weeks, at a dose of 60 mg/kg [28]; Group 4: diabetic rats that received C. colocynthis fruit pulp powder (100 mg/[kgd] divided into two doses) for 4 weeks orally by gavage; Group 5: diabetic rats that received C. colocynthis fruit pulp powder (300 mg/[kgd] divided into two doses) for 4 weeks orally by gavage; Group 6: diabetic rats that received topical C. colocynthis oil, five drops on the hind paw, twice a day for 4 weeks; Group 7: diabetic rats that received topical C. colocynthis ointment, 0.25 mg/kg on the hind paw, twice a day for 4 weeks. The drugs were administered for four weeks starting from the fourth week after STZ injection following previous studies [29,30]. For oral use, C. colocynthis fruit pulp powder and gabapentin were suspended in tap water to yield the concentrations described above, with a final volume of 10 mL. The medications were given via orogastric tubes. 2.11. Body weight and blood glucose level evaluation Body weight and blood glucose levels of rats were considered as metabolic parameters in the present study. Body weight was measured before and after the treatment, while blood glucose level was measured once after the injection of STZ or citrate buffer, and once after the treatment. 2.12. Sensation analysis Analgesic effects of C. colocynthis on STZ-induced neuropathy were measured by hotplate and tail-flick tests in treated and control rats. 2.12.1. Hot-plate test Hot-plate test is a physiologic test to evaluate the changes in the sensation of the paws of the animal. Following the method proposed by Eddy and Leimbach [29], rats were individually placed on a hot plate surrounded with a glass chamber with the temperature adjusted to 55–58 ºC. The latency of the first reaction such as licking, moving the paws, little leaps or a jump to escape the heat, was recorded. To avoid paw damage to the animals, they were not allowed to remain in the chamber longer than 10 s. The test was repeated three times within 30 min. The hot-plate apparatus, and analgesy-meter were purchased from Ugo Basile (Comerio, Italy).
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2.12.2. Tail-flick test Tail-flick test is a physiologic test to evaluate the changes in the sensation of the tail of the animal. A Socrel model DS 20 tail-flick apparatus (Apelex, Bagneux, France) was used to induce acute nociception. The tail-flick latency of the rats was determined as the time required for the rat to remove its tail from the thermal stimulus (induced by an intensity-controlled beam of light, on the distal one-third portion of the rat tail). The temperature was adjusted to 55–60 ºC and the cutoff time was fixed at 10 s to avoid tail injury. For each rat, two or three recordings were measured at an interval of 15 min, and the mean value was used for statistical analysis. 2.13. Biochemical antioxidant analysis 2.13.1. Blood and hemoglobin preparation for biochemical assay At the end of the study, the rats were fasted overnight with free access to drinking water. They were then sacrificed after anaesthetizing with ether (Merck Chemical Company, Germany). Blood samples were collected via heart puncture, and whole blood was transferred to sterile microtubes and centrifuged at 3000 × g for 5 min. Serum was collected in a new microtube and stored at –70 °C for assaying enzyme activity. 2.13.2. Antioxidant analysis To evaluate the protective effects of C. colocynthis on the STZ-induced oxidative damage in sciatic nerve tissue, antioxidant enzyme activity and malondialdehyde (MDA) were measured. Antioxidant enzymes include total superoxide dismutase (SOD) and catalase (CAT). The activity of total SOD was evaluated using Ransod Kits (Randox Company, UK), following the manufacturer’s instructions and previous research [31]. To evaluate CAT activity, we used a method described by Aebi [32]. The MDA level was evaluated using a modified HPLC method based on the reaction of MDA with thiobarbituric acid (TBA) to form a colored MDA-TBA adduct as previously described [22,33]. 2.14. Nerve histological analysis Immediately after collection of blood, sciatic nerves were removed and fixed in 10% formalin and processed in a tissue processor machine, and paraffin blocks were prepared. Microscopic sections (5 µm) were stained with hematoxylin and eosin and examined under a light microscope.
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2.15. Statistical analysis The results are expressed as mean ± standard error of the mean. All data were analyzed with the Statistical Package for Social Sciences (SPSS-16.0, IBM®). Analysis included one-way analysis of variance, followed by the Tukey test for post-hoc multiple comparisons among treatment groups. Statistical significance was set at P < 0.05.
3. Results 3.1. Qualitative analysis by TLC To qualitatively evaluate the CE content in C. colocynthis extract, TLC with ethylacetate:methanol (9:1) as the mobile phase was used. The presence of CE was affirmed by the retention factor (Rf value of 0.58) along with UV254 quenching properties. Likewise, after spraying the anisaldehyde/sulfuric acid reagent and heating, the bright yellow spot of CE was visualized. To identify pure CE compound within the C. colocynthis extract, a second spot was added to the TLC plate, the first belonging to the C. colocynthis extract, the second to CE standard, and ethylacetate:methanol (9:1) was used as the mobile phase. Careful monitoring found that the CE appeared with the same retention factor (Rf value of 0.58) as the C. colocynthis extract. These qualitative data confirmed the likely presence of CE in the extract of C. colocynthis fruit. 3.2. Reverse-phase HPLC quantitative analysis of CE using external standard In order to quantitatively analyze CE content, the mobile phase consisted of a gradient from 60:40 (v/v; water:methanol) to 100% methanol, with a flow rate of 1.0 mL/min, and detection was performed at a wavelength of 254 nm. The peaks for the CE standard are shown in Figs. 2a and 2b, respectively.
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Fig. 2. Reverse-phase high-performance liquid chromatography results. a: obtained from a standard for cucurbitacin E (tR 3.78 min) under optimized condition; b: obtained from cucurbitacin E in the extract of Citrullus colocynthis fruit.
The retention time (tR) of CE was (3.52 ± 0.08) min. The linear relationship between peak area and injected concentration fell in the range of 1 to 500 µg/mL (correlation coefficient R2 = 0.9989; Fig. 3). For three concentrations of the CE standard (1, 50 and 100 µg/mL), recovery (accuracy) of CE was measured to be 101.6%, 98.4% and 99.3%, respectively. The repeatability of retention time (RSD%) was calculated according to the interday and intraday comparison as 1.18% and 2.87%, respectively. Finally, analysis of C. colocynthis fruit pulp found that CE was present at (19.48 ± 1.31) mg/g of fruit pulp extract, and the recovery rate (recovery%) was (99.7 ± 0.84)%.
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Fig. 3. The linear relationship between concentration of cucurbitacin E (one of the most abundant chemical compounds isolated from Citrullus colocynthis fruit) and peak area fell between 1 and 500 µg/mL.
3.3. Total phenolic content and antioxidant analysis In the present study, we compared the antioxidant activity and total phenolic content of pulp and extracts of C. colocynthis. The extract of C. colocynthis had a strong DPPH radical-scavenging activity with an IC50 value of (11.76 ± 2.79) mg/mL, compared with the reference compound of pure quercetin, which had an IC50 of (4.62 ± 2.26) mg/mL. The pulp demonstrated an antioxidant activity with an IC50 value of (95.04 ± 2.21) mg/mL. The total phenolic content of the pulp and the extract of C. colocynthis was calculated from the regression equation from the calibration curve (Fig. 4, y = 0.008x + 0.197, R2 = 0.996). As expected, this extract showed a significantly higher total phenolic value ([27.97 ± 3.33] mg GAE/gE) than the pulp ([15.67 ± 1.40] mg GAE/gE). The findings of this analysis indicated that C. colocynthis had a high antioxidant activity which may be attributed to its phenolic contents.
Fig. 4. The calibration curve of gallic acid. Regression equation (y = ax + b).
3.4. Metabolic parameters: body weight and blood glucose
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After STZ injection, all rats showed high blood glucose (more than 300 mg/dL), indicating the induction of diabetes. The groups that received C. colocynthis, in any of its forms, had lower blood glucose than the diabetic control group (Fig. 5a). Orally administered C. colocynthis treatments had a better effect than topical ointment or oil treatments for decreasing blood glucose levels. Among oral C. colocynthis treatments, the 100 mg/kg dose was more effective than the 300 mg/kg (P < 0.0001). It appeared to have a greater glucose-lowering effect than gabapentin, but it was not statistically significant (P > 0.05). The group that received 100 mg/kg of C. colocynthis powder by oral gavage also exhibited better results in weight retention, relative to other C. colocynthis-treated groups (Fig. 5b). Although it was not as effective as gabapentin, the group that received 100 mg/kg C. colocynthis powder by oral gavage showed a significant effect (P < 0.0001) compared to the diabetic control group.
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Fig. 5. Effects of Citrullus colocynthis (Cc) on two metabolic parameters of test animals: blood glucose (a) and body weight (b). Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. ****P < 0.0001, ***P = 0.001, **P = 0.004, *P = 0.03.
3.5. Hot-plate test A significant decrease in response latency to heat stimuli in the diabetic control group, compared to the normal control group (P < 0.0001), affirmed the presence of DN (Fig. 6a). The reaction to heat was improved significantly in all C. colocynthis-treated groups: P < 0.01 for 300 mg/kg oral C. colocynthis and P < 0.0001 for others. The 100 mg/kg oral C. colocynthis treatment had the greatest treatment effect.
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Fig. 6. Effects of Citrullus colocynthis (Cc) on thermally-induced algesia, using the hot-plate model (a) and tail-flick model (b) in rats. Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. ****P < 0.0001, **P < 0.003.
3.6. Tail-flick test A significant decrease in tail-flick latency in the diabetic control group, compared to the control group (P < 0.0001), affirmed the presence of DN (Fig. 6b). The tail-flick latency was improved significantly in all C. colocynthis-treated groups (P < 0.0001), except 300 mg/kg oral C. colocynthis. In fact, oil and 100 mg/kg oral C. colocynthis exhibited even better recovery than gabapentin with no significant difference (P > 0.05). 3.7. Biochemical tests As shown in Fig. 7a, the SOD enzyme appeared to have a lower level in the diabetic rats compared to the normal control rats, but it was not statistically significant. CAT enzyme was decreased in diabetic rats as shown in Fig.7b (P < 0.0001). This was expected, as
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hyperglycemia-induced oxidative stress is strongly implicated in the pathogenesis of DN. Activity of these antioxidant enzymes was raised in the rats treated with gabapentin and C. colocynthis (except 300 mg/kg oral C. colocynthis), compared to those of the diabetic control group. The improvement of antioxidant enzyme activity was significant in the 100 mg/kg oral C. colocynthis group (P < 0.05 for SOD and P < 0.01 for CAT). The amounts of SOD and CAT in rats treated with oil and ointment were not significantly different from the diabetic control group.
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Fig. 7. Antioxidant effect of Citrullus colocynthis (Cc) evaluated with (a) superoxide dismutase (SOD), (b) catalase (CAT) and (c) malondialdehyde (MDA). Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. **** P < 0.0001, ** P < 0.01, * P < 0.02. Hb: hemoglobin.
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MDA level was elevated in the diabetic control group (Fig. 7c). The 100 mg/kg oral C. colocynthis group was the only test group that significantly affected MDA levels (P < 0.05), apart from the reference drug group. The latter appeared to have the greatest effect on MDA levels.
3.8. Histological evaluation Light microscopic investigations of the sciatic nerve of all rats were conducted. Rats from the normal control group had no changes in morphology of myelin and showed normal structure (Fig. 8a). Diabetic rats with no treatment showed demyelinated fibers and nerve degeneration (Fig. 8b). The numbers of demyelinated and degenerated sciatic nerve fibers were decreased in the rats treated with C. colocynthis and gabapentin, but there was no significant difference among them (Fig. 8c).
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Fig. 8. Results of microscopic section. a: microscopic section from sciatic nerve of control group showing normal nerve plexus with myelinated fibers (H&E stain, 100 ×). b: microscopic section from sciatic nerve of diabetic group showing degeneration and demyelination of nerve fibers (H&E stain, 400 ×). c: microscopic section from sciatic nerve of rats treated with Citrullus colocynthis showing less nerve fibers with degeneration and demyelination (H&E stain, 250 ×). H&E: hematoxylin and eosin.
3.9. Adverse effects There was intestinal bleeding in rats treated with 300 mg/kg oral C. colocynthis, which was detected after they were sacrificed. No other symptom of toxicity was seen in C. colocynthis-treated rats during the study.
4. Discussion
Based on the findings of the present study, C. colocynthis significantly improves physiologic, metabolic and pathologic features of neuropathy in diabetic rats. However, the four groups that received C. colocynthis products exhibited different results. This difference was not only dependent on the form, but also on the dose administered. Seven groups of rats (including the normal control and diabetic control) were followed in this study. Diabetic rats with no treatment, showed severe damage in all features (high blood glucose, impaired physiologic test, abnormal biochemical test and histology), compared to the control rats. Gabapentin-treated rats showed improvement in almost all tests compared to the diabetic rats with no treatment. Oral C. colocynthis treatment at a dose of 100 mg/kg had the best results among all C. colocynthis-treated rats. Administration of either gabapentin or 100 mg/kg C. colocynthis lead to significant positive outcomes compared to the diabetic rats with no treatment. On the other hand, oral C. colocynthis treatment at a dose of 300 mg/kg was less effective than all other treatments. The results for this group (except in glucose level and hot-plate test) were not significantly different from the diabetic control group. Oil and ointment, as topical treatments, had similar results in all tested parameters. They had their best effects in the thermal algesia tests and appeared to generally perform lesswell than other treatments. Oxidative stress plays an important role in the pathophysiology of DN [34,35] and its suppression can lead to amelioration of DN. The antioxidant capacity of C. colocynthis, shown in the present study, was in complete agreement with previous in vitro studies
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[36,37]. Antioxidant activity of C. colocynthis in diabetic rats has been investigated in three in vivo studies previously [38–40]. Dallak [38], Jeyanthi and Mary Violet Christy [40] used C. colocynthis pulp extract or seed in 300 mg/kg doses. Abd El-Bakey and Amin [39] used C. colocynthis fruit extract in a 50 mg/kg dose in their study. The results of their studies showed significant prevention of oxidative damage with C. colocynthis treatment. In contrast to these studies, the current work investigated the therapeutic, not preventive, effect of C. colocynthis, following the development of neuropathy and including a topical formulation, which is a routine, traditional use for neuropathy. The goal of DN treatment is sense improvement, since DN causes a symmetrical loss of distal sensation [41]. Thus, hot-plate and tail-flick tests were applied to evaluate heat sense in rats. The results showed the effectiveness of C. colocynthis on sense improvement in neuropathic rats. The metabolic evaluation was done in the present study by measuring the body weight and blood glucose level of the animals. C. colocynthis treatment (especially the oral dose of 100 mg/kg) appeared to be effective also in weight improvement of diabetic animals. Our results confirmed the blood glucose-lowering effect of C. colocynthis, not only in the oral form (which has been reported in many studies) but also in topical administration [42–44]. This makes C. colocynthis a good alternative therapy in DN, as its treatment is highly effective in glucose control. A similar decrease in blood glucose level was also effected by gabapentin, which agrees with previous reports [45,46]. The effect of C. colocynthis on DN was previously studied in a clinical trial [17]. It showed that topical application of C. colocynthis could decrease pain in patients with painful diabetic polyneuropathy. Our study confirmed that topical treatment was effective in decreasing neuropathy, according to the physiological and pathological findings. However, results showed that oral consumption might be more effective. According to TPM, C. colocynthis has a hot temperament of the fourth degree and dry of the second [47]. Also, the main cause of neuropathy, “owja’easab,” is considered to be the conversion of nerve temperament to cold [48,49]. Therefore, in TPM, it is recommended as a good choice in the treatment of neuropathy. Among C. colocynthis-treated groups, the best results were obtained at the 100 mg/kg oral dose and the worst were at the 300 mg/kg oral dose. Additionally, more adverse
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effects were observed in rats treated with 300 mg/kg dosage. Thus, determining the optimal dose is essential for effective treatment of neuropathy. The adverse event observed in the rats receiving the 300 mg/kg C. colocynthis dose was intestinal bleeding. Studies have reported colitis as an adverse effect of C. colocynthis [50]. Also, Javadzadeh et al. [51] reported four patients developing rectorrhagia after consuming C. colocynthis in a case series study. The pathophysiology of this adverse event is thought to be due to membranolytic activity of the saponins that are also contained in C. colocynthis [52]. The strength of the present study was that we evaluated the efficacy of different C. colocynthis preparations (topical as oil and ointment and oral powder), two dosages of the oral form of C. colocynthis; and these preparations were compared with the reference drug gabapentin in the treatment of DN. The limitation of the present study was the restriction of oral doses of C. colocynthis to 100 and 300 mg/kg. This work represents a preliminary study, and the dose effect that we found was not anticipated.
5. Conclusion
Results from the present animal study showed that C. colocynthis fruit, through its antioxidant and blood glucose-lowering activity, has a positive effect on the treatment of DN. Future studies should determine the optimum dosage of C. colocynthis in the treatment of DN, exploring the dose-response relationship. Biologically active components should be isolated from whole C. colocynthis extract to determine their individual contributions to the effects observed here.
Funding
This work was supported by the Shiraz University of Medical Sciences (grant number: 11222-01-01-95). The funding source had no role in study design, data collection, data analysis, data interpretation, writing of the report, or submission of the report as an article for publication.
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Acknowledgements
This article is extracted from the thesis submitted to the School of Medicine (Shiraz University of Medical Sciences, Shiraz, Iran) by Mohadeseh Ostovar, for fulfillment of PhD degree in TPM.
Conflict of interest
The authors declare no conflict of interest.
References
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S2095-4964(19)30117-7 https://doi.org/10.1016/j.joim.2019.12.002 JOIM 133
To appear in:
Journal of Integrative Medicine
Received Date: Accepted Date:
27 December 2018 20 May 2019
Please cite this article as: M. Ostovar, A. Akbari, M.H. Anbardar, A. Iraji, M. Salmanpour, S.H. Ghoran, M. Heydari, M. Shams, Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy, Journal of Integrative Medicine (2019), doi: https://doi.org/10.1016/j.joim.2019.12.002
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© 2019 Shanghai Changhai Hospital. Published by ELSEVIER B.V.
JIM-12-2018-OA-ER-0398 Original Research Article
Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy Mohadeseh Ostovar1, Abolfazl Akbari2, Mohammad Hossein Anbardar3, Aida Iraji4, Mohsen Salmanpour5, Salar Hafez Ghoran6, Mojtaba Heydari7, Mesbah Shams8
1. Department of Traditional Persian Medicine, School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 2. Department of Physiology, School of Veterinary Medicine, Shiraz University, Shiraz 73, Iran 3. Department of Pathology, School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 4. Central Research Laboratory, Shiraz University of Medical Sciences, Shiraz 73, Iran 5. Department of Pharmaceutics, Center for Nanotechnology in Drug Delivery, Pharmacy School, Shiraz University of Medical Sciences, Shiraz 73, Iran 6. Department of Chemistry, Golestan University, Gorgan 49, Iran 7. Research Center for Traditional Medicine and History of Medicine, Iran School of Medicine, Shiraz University of Medical Sciences, Shiraz 73, Iran 8. Endocrine and Metabolism Research Center, Shiraz University of Medical Sciences, Shiraz 73, Iran
ABSTRACT
Objective: This study investigated the biochemical, histopathological and physiological effects of Citrullus colocynthis on peripheral neuropathy in rats with streptozotocin (STZ)-induced diabetes. Methods: Seventy adult male Sprague-Dawley rats were included in the present study. Diabetes was induced in 60 rats, with a single intraperitoneal injection of STZ (65
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mg/kg). After 4 weeks, the diabetic rats were assessed for neuropathy. Then, the diabetic rats with neuropathy were randomly divided into 6 groups for a 4-week treatment with gabapentin, oral administration of C. colocynthis fruit pulp powder (100 and 300 mg/kg per day), topical preparations as oil-based solution and ointment, or placebo. Changes in metabolic, physiological, biochemical and histological parameters were considered as treatment outcomes. Results: Metabolic outcomes (body weight and blood glucose level) were improved in the C. colocynthis-treated groups as compared to placebo. Tail-flick and hot-plate tests also had lower latency in the C. colocynthis-treated groups. Measurement of oxidative stress markers (malondialdehyde, superoxide dismutase and catalase) showed the antioxidant effect of C. colocynthis. Histological evaluation of the sciatic nerve showed that C. colocynthis decreased the number of demyelinated and degenerated nerve fibers. Among the C. colocynthis-treated groups, the one receiving 100 mg/kg per day oral powder had the best treatment outcomes. Conclusion: The present study showed that C. colocynthis fruit, through its antioxidant and hypoglycemic activities, has a positive effect in the treatment of diabetic neuropathy. Please cite this article as: Ostovar M, Akbari A, Anbardar MH, Iraji A, Salmanpour M, Ghoran SH, Heydari M, Shams M. Effects of Citrullus colocynthis L. in a rat model of diabetic neuropathy. J Integr Med. 2019; Epub ahead of print. Received December 27, 2018; accepted May 20, 2019.
Keywords: Citrullus colocynthis; Diabetic neuropathy; Diabetes mellitus; Herbal medicine; Natural medicine; Traditional Persian medicine
Correspondence: Mesbah Shams, MD; E-mail address: [email protected]
1. Introduction
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Diabetes mellitus (DM) is a metabolic disease caused by problems in production and/or use of insulin and is diagnosed by high level of blood glucose. It is one of the most common chronic medical conditions encountered in clinical practice. The global prevalence of DM is known to be rapidly increasing as a result of aging populations and lifestyle changes. The International Diabetes Federation estimated that 415 million adults had diabetes in 2015 and the number would increase to 642 million by 2040 [1]. DM can lead to serious complications affecting multiple organs such as heart, blood vessels, nerves, eyes and kidneys. Diabetic neuropathy (DN), as a chronic microvascular complication of diabetes, results in significant morbidity and mortality [2]. About half of people with diabetes suffer from neuropathy [3]. The most common type of DN is distal symmetrical sensory neuropathy, or known in polyneuropathy. Studies on the etiology of DN have identified several biochemical mechanisms of nerve and neurovascular damage, among which, oxidative stress is thought to be the most important [4]. Both chronic and acute hyperglycemia cause oxidative stress in the peripheral nervous system, leading to an acceleration of DN development [5]. Due to the limited efficacy of the drugs commonly used to treat DN pain, phytotherapy is a common alternative therapy and is a popular treatment (amongst different complementary and alternative medicines) for many chronic diseases and their complications [6–8]. Citrullus colocynthis L. (Cucurbitaceae), also known as bitter apple, a well-known plant of ancient medical science, is a desert plant found in Africa, the Middle East and Asia [9]. In Iran, it grows in the southwest, southeast, central and eastern regions [10]. In traditional Persian medicine (TPM) references, such as Liber Continens written by Rhazes (865–925 A.D.) [11], Canon of Medicine by Avicenna (980–1037 A.D.) [12], and the Storehouse of Medicaments by Aghili Shirazi (1670– 1747 A.D.) [13], C. colocynthis is prescribed for patients with nerve-originating pain [14]. Recent studies have investigated many pharmacological effects of C. colocynthis, including antimicrobial, anti-inflammatory, antioxidant, analgesic, local anesthetic, hypolipidemic and blood glucose-lowering effect [15]. Also, its positive effect on diabetes and diabetic nephropathy, as one of its main complications, has been reported [16]. Moreover, we have recently reported the clinical efficacy of the topical use of C. colocynthis in patients with painful DN, showing its efficacy for decreasing pain and
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improving nerve function [17]. However, the underlying mechanism of its protective effect in DN is not well studied. This study investigates the underlying mechanism of the neuroprotective effect of C. colocynthis (considering the physiologic, biochemical, oxidative stress and pathologic outcomes) on DN in an animal model.
2. Material and methods
2.1. Ethical considerations All experimental protocols were approved by the institutional animal ethics committee of the Shiraz University of Medical Sciences, Shiraz, Iran (approval No. IR.sums.REC.1396.S12). Also, the recommendations of the European Council Directive (86/609/EEC) of November 24, 1986, regarding the standards in the protection of animals used for experimental purposes were followed. 2.2. Plant material Dried C. colocynthis fruits were purchased from a medicinal plant market in Shiraz; they were from a batch that had been collected in the Kazerun area of Southern Iran. Following authenticated by Dr. M. M. Zarshenas (Department of Phytopharmaceuticals, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran), a voucher number was specified for the sample (No. PM776). The sample was preserved in the herbarium of Pharmacy School at Shiraz University of Medical Sciences. 2.3. C. colocynthis formulations In order to evaluate the role of preparation on therapeutic efficacy, three types of C. colocynthis-containing products were prepared. 2.3.1. Powder of fruit pulp The pulp of dried C. colocynthis fruits was ground in a mechanical grinder. The resulting powder was sieved using # 50 mesh and kept in a freezer. 2.3.2. Oil-based solution and ointment The milled, dried C. colocynthis fruits were gently stirred with distilled water for 10 min. The mixture was concentrated using a rotary evaporator and then freeze-dried to obtain a fine dry powder. Then the extract was used to prepare oil-based solution and ointment as follows [18]. 2.3.2.1. Oil-based solution
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The extract of C. colocynthis fruits was boiled in sesame oil at 100 oC for 15 min, as traditionally recommended, followed by evaporation method to remove the water portion [18]. 2.3.2.2. Ointment The extract of C. colocynthis fruits was reconstituted in distilled water and emulsified in lanoline as 5% (w/w) product using soybean lecithin emulsifier. 2.4. Qualitative analysis by thin-layer chromatography Cucurbitacin E (CE), as one of the most abundant chemical compounds isolated from C. colocynthis fruit, was used as the reference compound (Sigma Chemical Co., > 98% purity; St. Louis, Mo, USA) [19]. In order to detect CE in the extract of C. colocynthis fruit, thin-layer chromatography (TLC) was performed on normal phase silica gel G254 plates (Merck, Germany). First, standard concentrations of CE (1 mg/mL) and C. colocynthis extract (10 mg/mL) were prepared. Having carefully examined various solvent systems, including n-hexane:ethylacetate, chloroform:acetone, chloroform:methanol and ethylacetate:methanol, the mixture of ethylacetate:methanol was selected to elute samples on the TLC sheet. Samples on the TLC sheets were detected under an ultraviolet (UV)254 lamp and visualized using anisaldehyde/sulfuric acid reagent followed by heating. All experiments were carried out in duplicate. 2.5. Quantitative analysis of CE using external standard 2.5.1. Reagents and instrumentation Standard CE was purchased from Sigma Aldrich Co. (high-performance liquid chromatography [HPLC] grade; ≥ 98% purity; St. Louis, Mo, USA). Methanol (HPLC grade) was purchased from Merck, Germany. High-purity water was prepared using Millipore purification system. The HPLC (KNAUER®) was equipped with quaternary K-1001 controller pumps and a K-2600 UV absorbance detector. The injector was matched with a 20 µL sample loop. 2.5.2. Reverse-phase HPLC condition The binary mobile phase consisted of 60% water and 40% methanol to reach the high polarity (100% methanol). The stainless steel analytical column used had the following properties [20]: Lichrosphere-100, reverse-phase C-18, 250 mm × 4.6 mm inner diameter, 5 µm, with precolumn. The UV-visible light detector was set at 254 nm as previously discussed [20]. Moreover, the flow rate and pressure were set at 1.0 mL/min
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and 190–220 bar, respectively. Having optimized the chromatographic conditions, all analyses were carried out at the room temperature. The volume of all injections into the column was 10 µL and the detecting took place in triplicate. 2.5.3. Preparation of standard CE solution, calibration curve and linearity About 1 mg of standard CE was precisely weighted and dissolved in 1 mL methanol (HPLC grade) to reach the final concentration of 1000 µg/mL. The calibration curve for CE was prepared using concentrations of 1 to 1000 µg/mL. The linear approximation was calculated for concentration range of 1 to 500 µg/mL. 2.5.4. Preparation of C. colocynthis extract About 1 g of the extract of C. colocynthis was weighed and dissolved in 50 mL methanol (HPLC grade). After sonication for 15 min, a cloudy solution was obtained. This solution was centrifuged and then filtered through Whitman quantitative filter paper (ashless, Grade 44, Merck, Germany) to yield the clear solution. 2.5.5. Estimation of CE from the extract of C. colocynthis The recorded area of all peaks was used for quantification of CE in the plant extract. In order to determine the amount of CE, the area values of each concentration were fitted to a calibration curve. 2.5.6. Precision and accuracy Precision was evaluated at two levels in order to verify the repeatability of retention time: interday and intraday. All experiments for this study were analyzed through five determinations at concentration of 5 µg/mL in one laboratory on one day. On the second day, the same samples were analyzed following the same methods. Outcomes were expressed in terms of standard deviation (SD) and repeatability SD (RSD%). The accuracy of the analytical method was expressed as recovery (%) and was measured by using standard addition method [21]. Three concentrations of standard (1, 50 and 100 µg/mL) were spiked into the same amount of examination samples (the prepared extract of C. colocynthis). 2.6. Antioxidant activity Analysis of the antioxidant activity of C. colocynthis was carried out according to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods, as previously described [22,23]. The antioxidant activity was determined by measurement of the decrease in the absorbance at 517 nm. Several aliquots, containing different sample concentrations, were tested and
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the results were reported by the percentage of residual or inhibited DPPH against the concentration of the sample. A stock solution of 110 μmol/L DPPH was prepared fresh by dissolving the appropriate amount of reagent in methanol. Twenty microliters of different concentrations of the sample extract or standard solution were mixed with 180 µL of a methanolic solution of DPPH and kept in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against methanol as blank. Percentage of DPPH reduction was determined using the following equation: percentage of DPPH reduction = (A0 – A1)/A0) × 100, where A0 is the absorbance of the control, and A1 is the absorbance in the presence of the sample. All determinations were performed in triplicate. 2.7. Determination of total phenolic content by Folin-Ciocalteu assay The total phenolic content of C. colocynthis was determined following the procedure of Waterhouse [24]. Diluted extract or blank liquid was mixed with 3.16 mL of water and 0.2 mL Folin-Ciocalteu reagent for 8 min at 22 °C. Then, 0.6 mL saturated sodium carbonate solution was added and the mixture was shaken vigorously for 20 s. The reaction was incubated in the dark at 22 °C for 2 h before measuring the absorbance at 765 nm using a UV-visible spectrometer (Cary 100 UV-Vis; Agilent Corporation, Santa Clara, CA, USA). A standard curve was prepared based on gallic acid standard solutions ranging from 0 to 1000 mg/L. The concentration of the total phenols was expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/gE). 2.8. Experimental animals Seventy healthy adult male Sprague-Dawley rats (Rattus norvegicus; [220 ± 15] g; aged 7–8 weeks) were colony-bred in the Animal House Center, Shiraz, Iran. SpragueDawley rat is a strain of albino rat used extensively in medical research because of its calmness and ease of handling. During the study period, standard pellet diet and water were given to experimental animals. They were housed (5 rats per cage) in the animal room under constant temperature at (22 ± 2) °C and humidity at (55 ± 5)%, with 12hour light and dark cycle and had free access to laboratory food and tap water. Special care was provided to minimize animal suffering and to reduce the number of animals used to the minimum required for statistical accuracy. Animals were acclimatized to
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laboratory conditions before the tests. All the experimental procedures were carried out between 09:00 a.m. and 09:15 a.m. 2.9. Induction and assessment of diabetes and neuropathy The diabetes was induced by a single intraperitoneal injection of a prepared solution of streptozotocin (STZ; 65 mg/kg body weight, Sigma-Aldrich, Germany) in 0.1 mol/L sodium citrate buffer (pH = 4.5); the control rats were injected with citrate buffer alone [25]. To prevent the drug-induced hypoglycemia, a solution of 5% glucose overnight was given to rats. To confirm diabetes, at the third day after STZ injection, the blood samples were collected via the rat tail vein technique using heparinized capillary glass tubes, and plasma glucose levels were measured by the enzymatic GOD-POD diagnostic kit method (Accurex®, India) [26]. The animals were considered diabetic if their blood glucose values were above 300 mg/dL. Then, after 4 weeks, neuropathy was confirmed using hot-plate test in the diabetic rats, by the Eddy and Leimbach method [27]. The animals were included in the study if the latency was more than 5 s; this latency indicates sensory dysfunction in rats. Fig. 1 shows the diagram of the experimental design.
Fig. 1. The diagram of the experimental design.
2.10. Experimental design and protocol To evaluate the effects of C. colocynthis in DN, the animals confirmed to have diabetes and neuropathy were divided into seven groups so that each group included ten rats as follows. Group 1: control rats or vehicle group, received a 0.1 mol/L citrate buffer (pH = 4.5) injection only at the start of the study and then distilled water orally by gavage
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for 4 weeks; Group 2: diabetic rats, as a diabetic control group, that received distilled water (0.05 mL twice a day) for 4 weeks orally by gavage; Group 3: diabetic rats, serving as a positive control, that received gabapentin daily by oral gavage, for 4 weeks, at a dose of 60 mg/kg [28]; Group 4: diabetic rats that received C. colocynthis fruit pulp powder (100 mg/[kgd] divided into two doses) for 4 weeks orally by gavage; Group 5: diabetic rats that received C. colocynthis fruit pulp powder (300 mg/[kgd] divided into two doses) for 4 weeks orally by gavage; Group 6: diabetic rats that received topical C. colocynthis oil, five drops on the hind paw, twice a day for 4 weeks; Group 7: diabetic rats that received topical C. colocynthis ointment, 0.25 mg/kg on the hind paw, twice a day for 4 weeks. The drugs were administered for four weeks starting from the fourth week after STZ injection following previous studies [29,30]. For oral use, C. colocynthis fruit pulp powder and gabapentin were suspended in tap water to yield the concentrations described above, with a final volume of 10 mL. The medications were given via orogastric tubes. 2.11. Body weight and blood glucose level evaluation Body weight and blood glucose levels of rats were considered as metabolic parameters in the present study. Body weight was measured before and after the treatment, while blood glucose level was measured once after the injection of STZ or citrate buffer, and once after the treatment. 2.12. Sensation analysis Analgesic effects of C. colocynthis on STZ-induced neuropathy were measured by hotplate and tail-flick tests in treated and control rats. 2.12.1. Hot-plate test Hot-plate test is a physiologic test to evaluate the changes in the sensation of the paws of the animal. Following the method proposed by Eddy and Leimbach [29], rats were individually placed on a hot plate surrounded with a glass chamber with the temperature adjusted to 55–58 ºC. The latency of the first reaction such as licking, moving the paws, little leaps or a jump to escape the heat, was recorded. To avoid paw damage to the animals, they were not allowed to remain in the chamber longer than 10 s. The test was repeated three times within 30 min. The hot-plate apparatus, and analgesy-meter were purchased from Ugo Basile (Comerio, Italy).
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2.12.2. Tail-flick test Tail-flick test is a physiologic test to evaluate the changes in the sensation of the tail of the animal. A Socrel model DS 20 tail-flick apparatus (Apelex, Bagneux, France) was used to induce acute nociception. The tail-flick latency of the rats was determined as the time required for the rat to remove its tail from the thermal stimulus (induced by an intensity-controlled beam of light, on the distal one-third portion of the rat tail). The temperature was adjusted to 55–60 ºC and the cutoff time was fixed at 10 s to avoid tail injury. For each rat, two or three recordings were measured at an interval of 15 min, and the mean value was used for statistical analysis. 2.13. Biochemical antioxidant analysis 2.13.1. Blood and hemoglobin preparation for biochemical assay At the end of the study, the rats were fasted overnight with free access to drinking water. They were then sacrificed after anaesthetizing with ether (Merck Chemical Company, Germany). Blood samples were collected via heart puncture, and whole blood was transferred to sterile microtubes and centrifuged at 3000 × g for 5 min. Serum was collected in a new microtube and stored at –70 °C for assaying enzyme activity. 2.13.2. Antioxidant analysis To evaluate the protective effects of C. colocynthis on the STZ-induced oxidative damage in sciatic nerve tissue, antioxidant enzyme activity and malondialdehyde (MDA) were measured. Antioxidant enzymes include total superoxide dismutase (SOD) and catalase (CAT). The activity of total SOD was evaluated using Ransod Kits (Randox Company, UK), following the manufacturer’s instructions and previous research [31]. To evaluate CAT activity, we used a method described by Aebi [32]. The MDA level was evaluated using a modified HPLC method based on the reaction of MDA with thiobarbituric acid (TBA) to form a colored MDA-TBA adduct as previously described [22,33]. 2.14. Nerve histological analysis Immediately after collection of blood, sciatic nerves were removed and fixed in 10% formalin and processed in a tissue processor machine, and paraffin blocks were prepared. Microscopic sections (5 µm) were stained with hematoxylin and eosin and examined under a light microscope.
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2.15. Statistical analysis The results are expressed as mean ± standard error of the mean. All data were analyzed with the Statistical Package for Social Sciences (SPSS-16.0, IBM®). Analysis included one-way analysis of variance, followed by the Tukey test for post-hoc multiple comparisons among treatment groups. Statistical significance was set at P < 0.05.
3. Results 3.1. Qualitative analysis by TLC To qualitatively evaluate the CE content in C. colocynthis extract, TLC with ethylacetate:methanol (9:1) as the mobile phase was used. The presence of CE was affirmed by the retention factor (Rf value of 0.58) along with UV254 quenching properties. Likewise, after spraying the anisaldehyde/sulfuric acid reagent and heating, the bright yellow spot of CE was visualized. To identify pure CE compound within the C. colocynthis extract, a second spot was added to the TLC plate, the first belonging to the C. colocynthis extract, the second to CE standard, and ethylacetate:methanol (9:1) was used as the mobile phase. Careful monitoring found that the CE appeared with the same retention factor (Rf value of 0.58) as the C. colocynthis extract. These qualitative data confirmed the likely presence of CE in the extract of C. colocynthis fruit. 3.2. Reverse-phase HPLC quantitative analysis of CE using external standard In order to quantitatively analyze CE content, the mobile phase consisted of a gradient from 60:40 (v/v; water:methanol) to 100% methanol, with a flow rate of 1.0 mL/min, and detection was performed at a wavelength of 254 nm. The peaks for the CE standard are shown in Figs. 2a and 2b, respectively.
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Fig. 2. Reverse-phase high-performance liquid chromatography results. a: obtained from a standard for cucurbitacin E (tR 3.78 min) under optimized condition; b: obtained from cucurbitacin E in the extract of Citrullus colocynthis fruit.
The retention time (tR) of CE was (3.52 ± 0.08) min. The linear relationship between peak area and injected concentration fell in the range of 1 to 500 µg/mL (correlation coefficient R2 = 0.9989; Fig. 3). For three concentrations of the CE standard (1, 50 and 100 µg/mL), recovery (accuracy) of CE was measured to be 101.6%, 98.4% and 99.3%, respectively. The repeatability of retention time (RSD%) was calculated according to the interday and intraday comparison as 1.18% and 2.87%, respectively. Finally, analysis of C. colocynthis fruit pulp found that CE was present at (19.48 ± 1.31) mg/g of fruit pulp extract, and the recovery rate (recovery%) was (99.7 ± 0.84)%.
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Fig. 3. The linear relationship between concentration of cucurbitacin E (one of the most abundant chemical compounds isolated from Citrullus colocynthis fruit) and peak area fell between 1 and 500 µg/mL.
3.3. Total phenolic content and antioxidant analysis In the present study, we compared the antioxidant activity and total phenolic content of pulp and extracts of C. colocynthis. The extract of C. colocynthis had a strong DPPH radical-scavenging activity with an IC50 value of (11.76 ± 2.79) mg/mL, compared with the reference compound of pure quercetin, which had an IC50 of (4.62 ± 2.26) mg/mL. The pulp demonstrated an antioxidant activity with an IC50 value of (95.04 ± 2.21) mg/mL. The total phenolic content of the pulp and the extract of C. colocynthis was calculated from the regression equation from the calibration curve (Fig. 4, y = 0.008x + 0.197, R2 = 0.996). As expected, this extract showed a significantly higher total phenolic value ([27.97 ± 3.33] mg GAE/gE) than the pulp ([15.67 ± 1.40] mg GAE/gE). The findings of this analysis indicated that C. colocynthis had a high antioxidant activity which may be attributed to its phenolic contents.
Fig. 4. The calibration curve of gallic acid. Regression equation (y = ax + b).
3.4. Metabolic parameters: body weight and blood glucose
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After STZ injection, all rats showed high blood glucose (more than 300 mg/dL), indicating the induction of diabetes. The groups that received C. colocynthis, in any of its forms, had lower blood glucose than the diabetic control group (Fig. 5a). Orally administered C. colocynthis treatments had a better effect than topical ointment or oil treatments for decreasing blood glucose levels. Among oral C. colocynthis treatments, the 100 mg/kg dose was more effective than the 300 mg/kg (P < 0.0001). It appeared to have a greater glucose-lowering effect than gabapentin, but it was not statistically significant (P > 0.05). The group that received 100 mg/kg of C. colocynthis powder by oral gavage also exhibited better results in weight retention, relative to other C. colocynthis-treated groups (Fig. 5b). Although it was not as effective as gabapentin, the group that received 100 mg/kg C. colocynthis powder by oral gavage showed a significant effect (P < 0.0001) compared to the diabetic control group.
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Fig. 5. Effects of Citrullus colocynthis (Cc) on two metabolic parameters of test animals: blood glucose (a) and body weight (b). Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. ****P < 0.0001, ***P = 0.001, **P = 0.004, *P = 0.03.
3.5. Hot-plate test A significant decrease in response latency to heat stimuli in the diabetic control group, compared to the normal control group (P < 0.0001), affirmed the presence of DN (Fig. 6a). The reaction to heat was improved significantly in all C. colocynthis-treated groups: P < 0.01 for 300 mg/kg oral C. colocynthis and P < 0.0001 for others. The 100 mg/kg oral C. colocynthis treatment had the greatest treatment effect.
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Fig. 6. Effects of Citrullus colocynthis (Cc) on thermally-induced algesia, using the hot-plate model (a) and tail-flick model (b) in rats. Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. ****P < 0.0001, **P < 0.003.
3.6. Tail-flick test A significant decrease in tail-flick latency in the diabetic control group, compared to the control group (P < 0.0001), affirmed the presence of DN (Fig. 6b). The tail-flick latency was improved significantly in all C. colocynthis-treated groups (P < 0.0001), except 300 mg/kg oral C. colocynthis. In fact, oil and 100 mg/kg oral C. colocynthis exhibited even better recovery than gabapentin with no significant difference (P > 0.05). 3.7. Biochemical tests As shown in Fig. 7a, the SOD enzyme appeared to have a lower level in the diabetic rats compared to the normal control rats, but it was not statistically significant. CAT enzyme was decreased in diabetic rats as shown in Fig.7b (P < 0.0001). This was expected, as
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hyperglycemia-induced oxidative stress is strongly implicated in the pathogenesis of DN. Activity of these antioxidant enzymes was raised in the rats treated with gabapentin and C. colocynthis (except 300 mg/kg oral C. colocynthis), compared to those of the diabetic control group. The improvement of antioxidant enzyme activity was significant in the 100 mg/kg oral C. colocynthis group (P < 0.05 for SOD and P < 0.01 for CAT). The amounts of SOD and CAT in rats treated with oil and ointment were not significantly different from the diabetic control group.
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Fig. 7. Antioxidant effect of Citrullus colocynthis (Cc) evaluated with (a) superoxide dismutase (SOD), (b) catalase (CAT) and (c) malondialdehyde (MDA). Error bars indicate 95% confidence interval, and the stars above the columns indicate the significance level of the pairwise comparison between each group and the diabetic control group. **** P < 0.0001, ** P < 0.01, * P < 0.02. Hb: hemoglobin.
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MDA level was elevated in the diabetic control group (Fig. 7c). The 100 mg/kg oral C. colocynthis group was the only test group that significantly affected MDA levels (P < 0.05), apart from the reference drug group. The latter appeared to have the greatest effect on MDA levels.
3.8. Histological evaluation Light microscopic investigations of the sciatic nerve of all rats were conducted. Rats from the normal control group had no changes in morphology of myelin and showed normal structure (Fig. 8a). Diabetic rats with no treatment showed demyelinated fibers and nerve degeneration (Fig. 8b). The numbers of demyelinated and degenerated sciatic nerve fibers were decreased in the rats treated with C. colocynthis and gabapentin, but there was no significant difference among them (Fig. 8c).
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Fig. 8. Results of microscopic section. a: microscopic section from sciatic nerve of control group showing normal nerve plexus with myelinated fibers (H&E stain, 100 ×). b: microscopic section from sciatic nerve of diabetic group showing degeneration and demyelination of nerve fibers (H&E stain, 400 ×). c: microscopic section from sciatic nerve of rats treated with Citrullus colocynthis showing less nerve fibers with degeneration and demyelination (H&E stain, 250 ×). H&E: hematoxylin and eosin.
3.9. Adverse effects There was intestinal bleeding in rats treated with 300 mg/kg oral C. colocynthis, which was detected after they were sacrificed. No other symptom of toxicity was seen in C. colocynthis-treated rats during the study.
4. Discussion
Based on the findings of the present study, C. colocynthis significantly improves physiologic, metabolic and pathologic features of neuropathy in diabetic rats. However, the four groups that received C. colocynthis products exhibited different results. This difference was not only dependent on the form, but also on the dose administered. Seven groups of rats (including the normal control and diabetic control) were followed in this study. Diabetic rats with no treatment, showed severe damage in all features (high blood glucose, impaired physiologic test, abnormal biochemical test and histology), compared to the control rats. Gabapentin-treated rats showed improvement in almost all tests compared to the diabetic rats with no treatment. Oral C. colocynthis treatment at a dose of 100 mg/kg had the best results among all C. colocynthis-treated rats. Administration of either gabapentin or 100 mg/kg C. colocynthis lead to significant positive outcomes compared to the diabetic rats with no treatment. On the other hand, oral C. colocynthis treatment at a dose of 300 mg/kg was less effective than all other treatments. The results for this group (except in glucose level and hot-plate test) were not significantly different from the diabetic control group. Oil and ointment, as topical treatments, had similar results in all tested parameters. They had their best effects in the thermal algesia tests and appeared to generally perform lesswell than other treatments. Oxidative stress plays an important role in the pathophysiology of DN [34,35] and its suppression can lead to amelioration of DN. The antioxidant capacity of C. colocynthis, shown in the present study, was in complete agreement with previous in vitro studies
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[36,37]. Antioxidant activity of C. colocynthis in diabetic rats has been investigated in three in vivo studies previously [38–40]. Dallak [38], Jeyanthi and Mary Violet Christy [40] used C. colocynthis pulp extract or seed in 300 mg/kg doses. Abd El-Bakey and Amin [39] used C. colocynthis fruit extract in a 50 mg/kg dose in their study. The results of their studies showed significant prevention of oxidative damage with C. colocynthis treatment. In contrast to these studies, the current work investigated the therapeutic, not preventive, effect of C. colocynthis, following the development of neuropathy and including a topical formulation, which is a routine, traditional use for neuropathy. The goal of DN treatment is sense improvement, since DN causes a symmetrical loss of distal sensation [41]. Thus, hot-plate and tail-flick tests were applied to evaluate heat sense in rats. The results showed the effectiveness of C. colocynthis on sense improvement in neuropathic rats. The metabolic evaluation was done in the present study by measuring the body weight and blood glucose level of the animals. C. colocynthis treatment (especially the oral dose of 100 mg/kg) appeared to be effective also in weight improvement of diabetic animals. Our results confirmed the blood glucose-lowering effect of C. colocynthis, not only in the oral form (which has been reported in many studies) but also in topical administration [42–44]. This makes C. colocynthis a good alternative therapy in DN, as its treatment is highly effective in glucose control. A similar decrease in blood glucose level was also effected by gabapentin, which agrees with previous reports [45,46]. The effect of C. colocynthis on DN was previously studied in a clinical trial [17]. It showed that topical application of C. colocynthis could decrease pain in patients with painful diabetic polyneuropathy. Our study confirmed that topical treatment was effective in decreasing neuropathy, according to the physiological and pathological findings. However, results showed that oral consumption might be more effective. According to TPM, C. colocynthis has a hot temperament of the fourth degree and dry of the second [47]. Also, the main cause of neuropathy, “owja’easab,” is considered to be the conversion of nerve temperament to cold [48,49]. Therefore, in TPM, it is recommended as a good choice in the treatment of neuropathy. Among C. colocynthis-treated groups, the best results were obtained at the 100 mg/kg oral dose and the worst were at the 300 mg/kg oral dose. Additionally, more adverse
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effects were observed in rats treated with 300 mg/kg dosage. Thus, determining the optimal dose is essential for effective treatment of neuropathy. The adverse event observed in the rats receiving the 300 mg/kg C. colocynthis dose was intestinal bleeding. Studies have reported colitis as an adverse effect of C. colocynthis [50]. Also, Javadzadeh et al. [51] reported four patients developing rectorrhagia after consuming C. colocynthis in a case series study. The pathophysiology of this adverse event is thought to be due to membranolytic activity of the saponins that are also contained in C. colocynthis [52]. The strength of the present study was that we evaluated the efficacy of different C. colocynthis preparations (topical as oil and ointment and oral powder), two dosages of the oral form of C. colocynthis; and these preparations were compared with the reference drug gabapentin in the treatment of DN. The limitation of the present study was the restriction of oral doses of C. colocynthis to 100 and 300 mg/kg. This work represents a preliminary study, and the dose effect that we found was not anticipated.
5. Conclusion
Results from the present animal study showed that C. colocynthis fruit, through its antioxidant and blood glucose-lowering activity, has a positive effect on the treatment of DN. Future studies should determine the optimum dosage of C. colocynthis in the treatment of DN, exploring the dose-response relationship. Biologically active components should be isolated from whole C. colocynthis extract to determine their individual contributions to the effects observed here.
Funding
This work was supported by the Shiraz University of Medical Sciences (grant number: 11222-01-01-95). The funding source had no role in study design, data collection, data analysis, data interpretation, writing of the report, or submission of the report as an article for publication.
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Acknowledgements
This article is extracted from the thesis submitted to the School of Medicine (Shiraz University of Medical Sciences, Shiraz, Iran) by Mohadeseh Ostovar, for fulfillment of PhD degree in TPM.
Conflict of interest
The authors declare no conflict of interest.
References
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