Effects of Aloe vera on colonic anastomoses of rats
Aydın I˙nan,
1* Meral S ¸ en,
1Cemile Koca,
2Metin Ergin
1and Cenap Dener
1Departments of1General Surgery and2Clinical Biochemistry, Fatih University School of Medicine, Ankara, Turkey.
Background: Aloe vera is a cactus-like plant that grows in hot, dry regions. Aloe vera has been known and used in traditional medicine and also in the modern world. Aloe vera has some pharmacological actions including wound healing, anti-inflammatory and immunostimulatory effects. We have studied the possible effects of Aloe vera gel on the healing of colonic anastomoses of rats.
Material and Methods: In this study, Sprague–Dawley rats were used. Aloe vera group received 2 mL/kg per day Aloe vera, and the control group received the same amount of water. Animals underwent resection and anastomosis of the distal colon. Bursting pressures of anastomoses and hydroxyproline contents of perianas- tomotic region were determined on the third and seventh days. Mann–Whitney U-test was used to compare bursting pressures and hydroxyproline levels between groups.
Results: There was no difference between groups in mortality. Mean bursting pressures and mean hydrox- yproline levels were lower in the control group than in the Aloe vera group both on the third and seventh days.
Conclusion: Aloe vera has multiple pharmacologically active compounds. It stimulates phagocyte formation and its activity induces nitric oxide production, it has angiogenic activity, increases synthesis, maturation and cross-linking of collagen, stimulates cell proliferation, stimulates fibroblast functions and proliferation, inhibits arachidonic acid oxidation, has anti-inflammatory effects, reduces tumour necrosis factor-a levels, and has antioxidant activity. Thus, according to our findings, Aloe vera has positive effects on the colonic anastomotic healing of rats.
Key words: Aloe vera, anastomotic healing, colon, rat.
Introduction
Aloe vera (Aloe barbadensis Miller) is a tropical cactus-like plant that grows readily in hot, dry regions of Africa, North America, Europe and Asia and belongs to the Liliaceal family, of which there are about 360 species.1–3 Aloe vera contains potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids.1
Aloe vera is known as the ‘healing plant’.4 It has been used in traditional medicine in several countries for many medical and cosmetic purposes and is said to be beneficial in the treatment of disorders such as arthritis, gout, acne, dermatitis and wounds such as peptic ulcers and thermal injuries and a broad range of illnesses.3–7The fresh gel, juice or formulated prod- ucts have been used.3
Aloe vera is also used to treat several health prob- lems in the modern world.4,6 Popular interest and use
of the Aloe vera gel have increased dramatically.
Harmful reactions due to Aloe gel treatment are recorded infrequently.8Currently, because of demand, Aloe vera, is cultivated in large quantities in several countries.1
Intestinal anastomotic leakage is still a serious surgical complication, which increases the hospital length of stay and perioperative morbidity and mortal- ity, although clinically significant leaks have a decreas- ing incidence.9,10Reported rates of clinical leakage of anastomoses are varying up to 30%.11Many indepen- dent local and systemic factors contribute to anasto- motic leakage.9
Healing of an anastomosis, like other wounds, is characterized by a series of events that involve a cascade of cellular and biological activities resulting in the restoration of tissue integrity and includes events characterized by inflammation, proliferation, collagen accumulation, and reorganization.12
As Aloe vera gel use has increased dramatically and has several important properties such as anti- inflammation and immunomodulation4,7,13 we studied the effects of Aloe vera gel on the healing of colonic anastomosis in the rats.
*Author to whom all correspondence should be addressed.
Email: aydininan@ttnet.net.tr
Received 11 May 2006; accepted 8 June 2006.
Materials and methods Ethical considerations
The Ethical Committee of Fatih University Medical School approved the study protocol.
Animals
Twenty-four male Sprague–Dawley rats (weighing 220–280 g) were used for this experimental study.
Rats were housed in an animal care facility with 12 h light–dark cycles, 21°C temperature and controlled humidity. Food (Medas¸ Sag˘ lık Ltd. S¸ ti, Ankara, Turkey) and water were given ad libitum.
Groups
Rats were assigned randomly to the Aloe vera group or the control group. The Aloe vera group received 2 mL/kg per day Aloe vera (Aloe Vera Life Essence Jel;
LR Kozmetik, Bursa, Turkey) for 2 weeks before surgery by gastric gavage while the control group received the same amount of water by the same method.
Anaesthesia
Anaesthesia was maintained by 30 mg/kg ketamine (Ketalar; EIS, Eczacıbas¸ı, Kirklareli, Turkey) intramus- cularly after a 12 h fast.
Operative procedure
Anaesthesia was maintained and the abdomen was shaved. After preparation with povidone-iodine the abdomen was draped using sterile technique. Steril- ized equipment was used for each animal. The peri- toneal cavity was entered through a 2 cm lower midline laparotomy. End-to-end inverted anastomosis was performed using eight to 10 interrupted 6/0 polypropylene (Prolene; Ethicon, Ankara, Turkey) sutures after resection of a 0.5 cm segment of the distal colon without damaging the mesenteric vascular arcade. The fascia was closed with running 3/0 polyglactin (Polysorb; Tyco Healthcare, Istanbul, Turkey) sutures, and the skin was closed with continu- ous subcuticular 4/0 polyglactin (Polysorb; Tyco Healthcare) sutures. Before the abdomen was closed, 1 mL of 0.9% NaCl was given into the abdomen as fluid resuscitation.
Postoperative period
Aloe vera group received 2 mL/kg per day Aloe vera by gastric gavage and the control group received the same amount of water by the same method during the
postoperative period as before. As bursting pressure of the anastomosis and hydroxyproline content of the perianastomotic tissue are measures of anastomotic healing,14–17 we have used these two parameters to evaluate anastomotic healing.
Bursting pressure
On the third day of the surgery, six rats from each group were separated randomly. After anaesthesia was maintained with ketamine (30 mg/kg), the perito- neal cavity was reopened. The entire colon was resected carefully. During the resection, great care was taken not to break down adhesions and wide resections were performed when necessary. Killing of animals was carried out with cardiac puncture after resection of the colon. The proximal end of the colon was connected to a continuous pressure monitoring system with a pressure transducer (KMA365B; Petas¸, Ankara, Turkey). The distal end was connected to a pump that supplies air at 1 mL/min and the entire colon was immersed under water. The pressure was recorded as ‘bursting pressure’ when air bubbles or a drop in pressure was seen. The assessor was blinded to the treatment given to the animals at the time of bursting pressure measurement.
Hydroxyproline determination
Following the measurement of bursting pressure, the colon was dissected along the mesenteric border.
Perianastomotic adhesions were carefully dissected.
Proximal 0.5 cm and distal 0.5 cm to the anastomotic line was resected for hydroxyproline determination of the anastomotic region. The resected colonic segment was stored at-80°C until further processing. Hydrox- yproline determination was performed using the tech- nique explained by Jamall et al.18
On the seventh day, the same interventions were performed on the remaining rats of each group. Due to the death of two rats, the number of rats in each group was five on the seventh day.
Statistical analysis was performed using SPSS for Windows® 10.0. The Mann–Whitney U-test was per- formed to determine statistical differences between bursting pressures and hydroxyproline levels of peri- anastomotic regions of Aloe vera and control groups.
Statistical significance level was set at P< 0.05.
Results
A rat from the control group on the fourth day of surgery and a rat from the Aloe vera group on the fifth day of surgery died. These deaths occurred because of septic complications of the anastomotic leakage.
There were no surgical or anaesthetic complications other than these deaths. There was no statistical dif- ference between groups regarding mortality (P> 0.05). All other animals survived until they were killed.
Bursting pressure indicates the mechanical strength of the anastomosis. Mean bursting pressure of the control group (3rd day: 84.00⫾ 27.31 mmHg, 7th day:
196.83⫾ 24.28 mmHg) was lower than the Aloe vera group (3rd day: 99.33⫾ 46.28 mmHg, 7th day:
244.00⫾ 20.63 mmHg) both on the third and seventh days. But it was only statistically significant on the seventh day (3rd day: P= 0.583, 7th day: P = 0.011).
Hydroxyproline content indicates the collagen accu- mulation in the anastomotic region. Mean hydroxypro- line content of the control group (3rd day: 0.231⫾ 0.073mg/mg tissue, 7th day: 0.341⫾ 0.063 mg/mg tissue) was lower than the Aloe vera group (3rd day:
0.404⫾ 0.078 mg/mg tissue, 7th day: 0.539 ⫾ 0.052mg/mg tissue) both on the third and seventh days. These results were significant on both days (3rd day: P= 0.006, 7th day: P = 0.028).
Discussion
Aloe vera has been one of the most widely used healing plants used throughout history and is used for medicinal purposes in several cultures.1,19,20Aloe vera is also cultivated in Turkey.7
Multiple pharmacologically active compounds are present in the Aloe vera plant.21These plants contain two major materials. Yellow exudates contain a high concentration of anthraquinone compounds and a clear mucilaginous gel.1,5,20
Reports on the beneficial effects of Aloe vera for a variety of conditions are available. These reports deal with radiation dermatitis, chronic leg ulcers, burns, frostbite, wounds and other skin lesions, soft tissue injuries, antiseptic effects, antimicrobial, antiviral, anti- fungal, and antiparasite actions, immunostimulatory, anticarcinogenic and antileukaemic activity, peptic ulcer, other gastrointestinal disorders, antidiabetic and hypoglycaemic effects, cardiac stimulatory effects, cholesterol-lowering effects, laxative effects, anti- inflammatory activity, arthritis, antioxidative, oedema, analgesic activity, gout, asthma, cosmetic purposes, general health, and effects on liver function.1,3,6–8,13,19–25
Aloe vera ingestion exerts some beneficial effects on various age-related diseases and significantly sup- presses multiple causes of death in male Fisher rats.
Aloe vera may have some beneficial effects on the progression of chronic nephropathy and may be useful in the prevention of thrombus formation.6
Wound healing is a complex and systematic process that requires a variety of cellular activities.
Phagocytosis, chemotaxis, mitogenesis and the syn- thesis of matrix components take place. The extracel- lular matrix influences cell migration, proliferation, adhesion and differentiation, and regulates cellular functions during immune responses with its adhesion receptors. Collagen is the major protein of the extra- cellular matrix. Synthesis and cross-linking of collagen and remodelling take place during the healing pro- cess.3Healing of gastrointestinal anastomoses follows almost the same series of events and requires many processes and factors.12,26,27
Aloe vera has been known to promote wound healing. However, the exact mechanism of its healing activity is still unknown and may have several activi- ties.8,19 According to our findings, Aloe vera has a positive impact for anastomotic healing of the colon.
Although the exact mechanism cannot be clarified, the effects of various active constituents of Aloe vera may explain some of these findings.
Aloe vera stimulates phagocyte formation and activity.8Activation of macrophages enhances wound healing. Macrophages function as phagocytes and debridement agents. They produce growth factors that influence stages of wound repair. Aloe compo- nents activate macrophages and stimulate T cells and induce nitric oxide production.20 Nitric oxide has been shown to play an important role in the optimal healing of colonic anastomoses by several mechanisms.12
Angiogenesis is required during wound healing.
Angiogenesis supplies oxygen and metabolites to new tissues, and disposes the waste products of metabo- lism during the wound repair. Angiogenesis might even be a key regulating process in order to heal wounds, because if angiogenesis is impaired, wound healing is retarded or unsuccessful.19Angiogenesis is also necessary in the healing process of the colorectal anastomosis because adequate tissue vasculariza- tion, perfusion and oxygen delivery are important factors for anastomotic healing.28 Some angiogenic activators have been shown to promote wound healing. Aloe vera gel actively induces the formation of new blood vessels.4,8,19b-Sitosterol is the major active component of Aloe vera gel that has the angiogenic activity.19 b-Sitosterol also has hypocholesterolaemic and hypotriglyceridaemic effects in experimental ani- mals.29Aloe vera also reduces oedema formation and increases oxygen access as a result of increased blood supply.5,8,19
There is a rapid increase in the synthesis of collagen in a healing wound. Collagen provides strength and integrity. Treatment of wounds with Aloe vera gel either
by oral or topical treatment increases the maximum levels of collagen in the granulation tissue.3,8,22In the present study, hydroxyproline levels of Aloe vera- treated rats were higher both on the third and the seventh days. This shows that the collagen content of the perianastomotic region of Aloe vera-treated rats was higher than in controls. Aloe vera also provides rapid maturation of collagen in addition to stimulation of collagen synthesis.3,8The collagen/DNA ratio of the granulation tissues of the wounds also suggests an increase in the synthesis of collagen per cell of treated wounds.8,22,30
Aloe vera extract enhances glycosaminoglycan in the wound matrix.30The whole extracts or components of Aloe vera stimulate cell proliferation with unknown mechanisms4,22,31 and stimulate protein synthesis.32 Aloe vera-treated wounds include higher protein and DNA content than untreated wounds.8,22
Cross-linking to form fibres follows the synthesis of collagen molecules. Remodelling of collagen and the formation of cross-links give rise to wound strength. As treated wounds show greater tensile strength, Aloe vera gel is thought to not only increase collagen syn- thesis per cell, but also aid in cross-linking.3,22 The collagen obtained from Aloe-treated wounds has a higher content of aldehydic groups than collagen from untreated controls. This may indicate that the collagen in treated wounds may undergo a higher degree of cross-linking, resulting in an ultimate increase in wound strength. Highly cross-linked col- lagen becomes less soluble in neutral buffer and acid solutions. Aloe vera treatment decreases solubility in neutral buffer and in acid solution of the collagen of the granulation tissue.3 Our study confirmed these find- ings as bursting pressures- which show the mechani- cal strength of anastomosis- of Aloe vera-treated rats were higher than controls.
Aloe vera gel is thought to contain growth factors that enhance the breaking strength of wounds.8Aloe vera gel provides an increased rate of wound contrac- tion and leads to quicker healing.3,22Aloe vera gel also stimulates fibroblast functions3,4,22 and prolifera- tion.3,8,23 A glycoprotein fraction of Aloe vera signifi- cantly stimulates cell proliferation, accelerates the recovery of a wound, and increases the expression of epidermal growth factor (EGF) receptor, fibronectin, and fibronectin receptor.4 EGF and fibronectin both play roles in wound healing.33As well as the complete extract of Aloe vera, its components aloesin, mannose-6-phosphate, glycoprotein and aloe-emedin have been reported to stimulate cell proliferation. It is possible that Aloe vera contains many cell proliferation-stimulatory components with varying activities.4
The blood supply is dependent on the formation of new blood vessels in anastomosis. Fibronectin, syn- thesized and secreted by fibroblasts and endothelial cells, serves as a connecting agent for the migrating endothelial cells.28
Aloe vera inhibits arachidonic acid oxidation,8,31has antiprostaglandin activity against thromboxane A223,32
and reduces thromboxane B2levels.31,32Aloe vera also decreases prostaglandin (PG) F2 alpha.32One of the active compounds in the Aloe vera plant is salicylic acid, which can be converted into a salicylate and thereby inhibit PG synthesis. Another active sub- stance, magnesium lactate, can inhibit histadine decarboxylase and thus acts as an antihistamine.21,32 The anti-inflammatory effect of Aloe vera is related to cyclooxygenase inhibition, rather than an effect on lypooxygenase activity. The inhibition of PGE2 formation by the extract may account for the anti- inflammatory effects, as PGE2 is the main prostaglan- din in the inflammatory exudate.5 Aloe vera controls and heals acute inflammation, as distinct from chronic inflammation, which was observed following gel treat- ment of excision and incision wounds in rats.8
Daily treatment of Aloe vera can reduce both tumour necrosis factor (TNF)-a and interleukin (IL)-6 levels, which might play an important role in anti-inflammation and growth promotion.13TNF-a inhibits collagen gene expression and increases collagenase activity, both of which decrease anastomotic strength. Decreased TNF-a levels increase anastomotic bursting strength in a rat model of peritonitis.34
Aloe vera has antioxidant activity and protects pro- oxidant-induced membrane and cellular damage by a significant reduction in the levels of cytochrome P450
and cytochrome b5.2
Aloe vera has beneficial effects on the cutaneous microcirculation.30Aloe vera also has vasodilator func- tion.35But there is no data showing whether Aloe vera has any effect on mesenteric microcirculation, based on a Medline search.
Aloe vera has antibacterial and antifungal proper- ties.21,32,35 Aloe vera gel eliminates common wound pathogenic bacteria, particularly Pseudomonas aeruginosa.23,30 Antibacterial properties of Aloe vera include Mycobacterium tuberculosis, Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Salmonella paratyphi, Streptococcus agalactiae, Enterobacter cloacae, Citrobacter species, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Streptococcus faecalis and Candida albicans in a dose- and concentration-dependent manner in both in vivo and in vitro studies.32
Studies with Aloe vera in mice revealed no acute toxicity in therapeutic doses.1 It has been observed
that long-term Aloe vera ingestion has no harmful effects, cause no change in physiological parameters, and lightens the disease burden during the ageing process in rats.6
In conclusion, Aloe vera has positive effects on the colonic anastomotic healing of rats according to our findings. Several explained actions of Aloe vera may play a role in this process. As Aloe vera has been widely used, future studies are needed to explain the exact mechanisms of the effects of Aloe vera on anas- tomotic healing and the effects of this plant should be studied in detail.
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