Toxicology Mechanisms and Methods, 19:73–77, 2009Copyright ©c Informa UK Ltd.ISSN: 1537-6516 print; 1537-6524 onlineDOI: 10.1080/15376510802442444

Influence of Aloe Vera Gel on Dermal Wound HealingProcess in Rat

Nasrin TakzareDepartment of Anatomy,School of Medicine, TehranUniversity of Medical Sciences,Tehran, Iran

Mir-jamal HosseiniDepartments of Toxicology andPharmacology, School ofPharmacy, Zanjan University ofMedical Sciences, Iran

Gholamreza HasanzadehDepartment of Anatomy,School of Medicine, TehranUniversity of Medical Sciences,Tehran, Iran

Hamideh MortazaviDepartment of Midwifery,School of Nursing andMidwifery, Tehran University ofMedical Sciences, Tehran, Iran

Alireza TakzareDepartment of Anatomy,School of Medicine, TehranUniversity of Medical Sciences,Tehran, Iran

Parisa HabibiDepartment of Animal Biology,School of Biology, College ofScience, University of Tehran,Iran

ABSTRACT In this topical study the influence of Aloe Vera, on the woundhealing process was investigated in 63 male rats with microscopic and cellcount methods. On the day of surgery a round wound, of diameter 20 mm, wascreated on the back of rats necks under sterile conditions. The surgery day wasdetermined as day zero (0). Then the rats were divided randomly into controland experimental groups 1 and 2. Animals in each group were sub-divided tothree smaller groups, investigated every 4, 7, and 14 days. From day 0, woundsurfaces were covered with gel once daily in experimental group 1 and twicedaily, for 12 h interval, in experimental group 2. Each rat received 30 g of thegel. The wound surface and healing were assessed on days 4, 7, and 14, andthen a sample from the wound was prepared and investigated microscopically.The results show that the number of neutrophil, macrophage, and fibroblastcells and the wound thickness in the control group were statistically differentfrom the experimental groups. It was found that the wound diameter thicknessin the experimental group was greatly lower due to twice administration of geland the power of wound healing was more than other groups.

KEYWORDS Aloe Vera; Gel; Wound Healing; Natural Medicines; Rats; Microscopic

INTRODUCTIONThe aim of wound healing, in medical science is a short time for repair and with few side

effects. Acceleration in wound healing with an extra stimulation of a factor can decrease thetime of patient motionlessness and therapeutic costs. It causes rapid return of the woundedto their daily activities. Natural medicines, especially pharmaceutical herbs, have beenconsidered the principal cure for several years. Today, further tendency to pharmaceuticalherbs usage is made. These have been used in the treatment of a variety of disordersincluding wound and burns since ancient times.

Wound healing, a highly complex process, and in short time with less side effects,is one of the objectives of medical science (Chithra et al. 1998). Since the ancienttimes, Egyptian, Greek, Indian, and European physicians had been attempting to treatwounds in a shorter time with less side effects (Goutoid et al. 1996), concerning theimportance of wound healing and because infection and cancer occur in cases of nothealing the wound (Adzick 1996). Various researches have been done on wound healing,consequently different substances have been made as wound healing and introducedthat often are herbal compounds and sometimes are chemical ones (Somboonwong etal. 2000; Adzick 1996). But none of them can be offered as an effective medicine tillnow. The effects of Ca, Cu, Zn ions, ultra sound waves, and the effects of chemicalsubstances such as hydrocortisone, Vit A, Ascorbic Acid, phenition, salt serum, and growthfactors can be mentioned as some examples (Stanford and Rappole 1969; Barnett andVarly 1987; Brown and Mcdonnell 1988; Mantle et al. 2001; Kumar and Jagetiz 2005).

Received 14 July 2007;accepted 22 November 2007.Address correspondence to NasrinTakzare, Department of Anatomy,School of Medicine, Tehran Universityof Medical Sciences, PO Box:13145–784, Tehran, Iran. E-mail:takzare@yahoo.com;m hosseini@razi.tums.ac.ir

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Natural drugs, especially pharmaceutical herbs, were consideredthe principal treatment and even the only healing methodin some cases for several years and meanwhile the primarysubstance of them were used in the pharmacy industry (Mantleet al. 2001). Today further tendency to pharmaceutical herbsusage is made due to less side effects, effective compounds,and expansion-dependent industries to pharmaceutical herbscultivation, prevention from foreign exchange exit, job creation,and especially WHO recommendation for using pharmaceuticalherbs. Injured tissue healing is of highly complex homeostaticbody activities including a series of complicated and regularprocesses. Rate of wound healing is affected by various factorssuch as nutrition, vitamins, steroidal hormones, oxygen, andenvironmental factors (Brown and Mcdonnell 1988; Linchet al. 1989; Kagan and Warden 1994; Atiyeh Bishara et al. 2003).Aloe Vera is one of the pharmaceutical herbs belonging to theliliaceae family. It has been used in the treatment of a varietyof disorders including infections dermatologic conditions andused as a laxative since ancient times. This plant has long meatythick leaves with twisted sides which end in thorns (Grindlay andReynold 1980; Heggars et al. 1993). The substance inside the leafcalled gel consists of 99% water with long chain polysaccharide,of Acetylated glucoannan kind, and other carbohydrates. It alsocontains the complex of Amino Acids, salisilic Acid, AscorbicAcid, Vit A, and Vit E with anti-oxidant properties (Grindlayand Reynold 1980; Davis et al. 1988; Heggars et al. 1993;Chithra et al. 1998). This gel prevents skin dryness due tothe high volume of water. The high percentage of glucosepresent in gel prevents bacterial growth due to the high osmoticvirtue (Adzick 1996). Prostaglandin and bradykinin hydrolyzingenzymes in Aloe Vera reduce pain and inflammation. Theexistence of amilaze enzyme in Aloe annihilated the necrosaltissue, aloctin-A, which has a cell division and mitosis effect andcauses the acceleration in healing and stimulating macrophageto excrete the dead tissue. Amino Acids present in plant gelare used to produce protein, causing tissue growth and healing.Vitamins including β-carotene, Vit E, Vit C, and B complex,used in cell reaction, are consumed as antioxidants in strength-ening the body immune system (Vazquez et al. 1996). Plantjuice, antrakinons with anti-microbial, antiviral, antifungal, andanti-inflammatory properties and the saponins with antisepticproperties are effective in preventing infections (Brown andMcdonnell 1988; Vazquez et al. 1996; Kumar and Jagetiz 2005).

Concerning the above information, complementary researchto study the influence of different doses of Aloe Vera gel on thedermal wound healing process was performed.

MATERIALS AND METHODSExperimental Animals

In this experimental study, 63 male wistar rats, weighing200–250 g, 10–12 weeks old, were used. Rats were housedon a 12-h light and 12-h dark cycle with unrestricted accessto food and water. They were kept in individual cages in acontrolled room (temperature 20–25◦C; humidity 70–80%).The animal room was under standard conditions. All exper-iments were conducted in the Tehran University accordingto the recommendations of the ethics committee on animal’sexperimentation of medical school.

Wound CreationOn the surgery day, rats were anaesthetized. Ketamin

hydrochloride 5 mg/100 g body weight (Gedeonrichter Factory,German) and diazepam 0.45 mg/100 g body weight (KimidarooCompany, Iran) and pentazocin 0.04 mg/100 g body weight(Toliddaroo Company, Iran) were used as anesthetic drug. Allthe anesthetic drugs were injected intra muscularly. Under thegeneral anesthesia, the back of the rat’s necks were shavedand antisepticized with povidin–Iodine (Toliddaroo Company,Iran) under sterile conditions. From day 0, wound surfaces werecovered with gel once daily in experimental group 1 and twicedaily, for 12 h intervals, in experimental group 2. Each ratreceived 30 g of the gel.

The surgery day was determined as day zero (0) and thefollowing days were 1, 7, and 14. All wounds were of full-thickness type extending up to the adipose tissue. Excisionwounds of size 4 cm2 were made by cutting out 2 cm ∗ 2 coppiceof skin from the shaven area (a round full-thickness wound, ofdiameter 20 mm was made). The randomized method was usedto divide the rats in the control group and experimental groups1, 2. Animals in each group were sub-divided into three smallergroups and were evaluated every 4, 7, and 14 days. From dayzero, wound surfaces were covered with Aloe Vera gel once dailyin experimental group 1 and twice daily in experimental group2. Nothing was used on wounds in the control group.

On days 4, 7, and 14 after the treatments of rats inexperimental groups, animals were sacrificed by ether inhala-tion, then a sample from the wound matrix and healthyskin beside that was made, Then, after tissue processing andfixation (Formaldehyde 4%) and staining they were observedmicroscopically for histological changes.

Sacrificed animals on the 1st, 7th, and 14th days after woundcreation, and the entire wound on each animal was cut outand stored at −70◦C until analysis. All of the samples werecolored with the Hematoxylin-Eosin method. Ten parts of thewound were assessed by using the optic part of a photomicro-scope. In this evaluation, fibroblasts, macrophage, neutrophil,and vessels endothelium were counted.

Wound Healing Assessment MethodWound healing was assessed by various parameters such

as evaluation of wound surface, percentage of wound healing,duration of healing, and complete healing. Wound surface ondays 4, 7, and 14 after creation was evaluated, of cm2 unit,and the percentage of healing was normalized by the followingformula:

Percentage healing

= Wound surface on day 1 − wound surface on day x × 100Wound surface on day 1

where x is the day when wound surface is evaluated.

Preparation of Aloe Vera GelThe important matter in this study was preparation of the

pure and effective substance of Aloe Vera gel. According torecent studies pharmaceutical usage of Aloe Vera plant aredivided into two parts: Aloe Resins or Aloe Antrakinons, which

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is yellow and bitter and is placed on the outer surface ofAloe skin and consist of Aloin, Isobarbaloin, and chrysaphanicBarbaloine. These compounds are semi-toxic. The second partis Aloe gel, which is a substance in the inner part of the plantleaf and consists of water with a polysaccharide, of acetylatedglucomannan kind, and other carbohydrates. Also gel containsthe complex of amino acid, salicylic acid, Ascorbic Acid, Vit A,and Vit E with Antioxidant properties. Aloe leaves can activatethe macrophage and control cell immune system, fibroblasts,and granulocytes. In our study, leaves were just used so that thepure gel preparation needed specialty and direct supervision.

The gel preparation process was performed in some phases.At first, the healthy plant leaves of Aloe Vera (3 years) werecollected from Karaj farmland (West of Tehran, Iran), thenwere cleaned and antisepticized. In the next phase, full sizemature leaves were cut from the plant and the rind removed.But, due to the sensitivity of the investigation, the effectivefactors in wound healing abandoning the toxic compoundsof the plant gel extraction was prepared as incision (slice).Longitudinal incision was made on the plant leaf and thegel present in the leaf was extracted for gel preparation. Theextracted gel was heat at a temperature 50◦C and filtered to beused in formulation. To make the gel constant it was mixed withsome glycerin and triethanolamin and heated at a temperatureof 75◦C. After completing the mentioned phases, a gel of 95%purity was obtained.

Grouping of AnimalsAfter wound creation, experimental animals were divided

into three groups of 21 rats. Then each group was sub-dividedto three smaller groups, investigated every 4, 7, and 14 daysas follows: Control groups—wounds untreated, Experimentalgroup1—wound treated once daily, and Experimental group2—wound treated twice daily in 12 h interval.

Statistical AnalysisExperimental groups 1 and 2 were compared with the

control group. Student’s t-test used to identify differencesbetween groups, and data were considered significant atp < 0.05, p < 0.01 and p < 0.001. To count the wound cells, aphotomicroscope (×40) was used.

RESULTSThe results obtained from the study are shown in Fig. 1–5.

The results show that number of neutrophil, macrophage,fibroblast cells, and the wound thickness in the experimentalgroup had statistically significant differences. On the 4thday of the experiment, number of fibroblast, macrophageand neutrophil cells, and the vessel endothelium were sig-nificantly different between experimental and control groups(p < 0.0001).

The results on the 7th day demonstrated that number offibroblast, neutrophil and vessels endothelium, and woundthickness in the experimental and control groups were signifi-cantly different (p < 0.001).

The results on the 14th day showed that number offibroblast was significantly different between control groupand experimental group 1 (p < 0.001). However, there was

FIGURE 1 The number of macrophage of aloe vera treated anduntreated wound tissue in rats. Data are given as mean ± SD forseven animals in each group.

FIGURE 2 The number of neutrophil of aloe vera treated anduntreated wound tissue in rats. Data are given as mean ± SD forseven animals in each group.

FIGURE 3 The number of fibroblast of aloe vera treated anduntreated wound tissue in rats. Data are given as mean ± SD forseven animals in each group.

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FIGURE 4 The number of macrophage of aloe vera treated anduntreated wound tissue in rats. Data are given as mean ± SD forseven animals in each group.

FIGURE 5 The thickness of wound in aloe vera treated anduntreated wound tissue in rats. Data are given as mean ± SD forseven animals in each group.

no significant difference in the number of macrophage, Neu-trophil, and vessels endothelium between the control group andexperimental group 1.

No significant difference was found in wound thicknessbetween the control group and experimental group 1 on the14th day, but there was a significant difference between controlgroup and experimental group 2 (p < 0. 05). The mentioneddifference was found between experimental groups 1 and 2(p < 0.05).

DISCUSSIONAloe Vera, of the pharmaceutical herbs, has been used in the

treatment of many disorders including infections, dermatologiccondition, and used as a laxative since ancient times. Inthe present study, the influence of Aloe Vera gel on dermalwound healing is evaluated. The findings of the present studyconfirm the significant acceleration of dermal wound healingin rats following administration of gel twice daily. The resultof this study is similar to previous studies and shows that the

application of gel accelerates the process of tissue bud creationand wound healing.

Another study mentioned that wound healing is differentwhen Aloe Vera gel is administered, but the concentration ofconsumed Aloe Vera gel was not clear and it was mentionedthat various concentrations of gel can be different in woundhealing acceleration (Visuthikosol and Chowchuen 1995)

In the present study, the number of fibroblasts in bothexperimental groups is more than in the control group. Thisincrease is due to the significant acceleration in the healingprocess in experimental groups and the proliferation phase ofthe healing process has begun earlier during that the same day,and, in the experimental groups, the number of macrophages ismore than in the control group. Also the number of Neutrophilsin experimental group 2 is more than in the control andexperimental group 1. Wound diameter in experimental group2 was lower than the other two groups and this is due tothe increase in number of fibroblasts in experimental group2 compared to the control group. The role of these cells inextra cellular substance synthesis can be justified and the twiceapplication of gel during the day was more effective. In thesame researches (Atiyeh Bishara et al. 2003; Dill and Lacopino2004), in order to know the reason and effective mechanism ofAloe Vera gel, rate of wound healing is mentioned to be dueto the increase in collagen synthesis by fibroblasts. Aloe Veragel contains various compounds such as polysaccharides andspecial Amino Acids possessing power of stimulation (Crindlyand Reynolds 1986; Chithra et al. 1998).

After administration of gel on the wound, the number offibroblasts increased compared to the control group and thisincrease caused the creation of tissue bud with excess rate.Also the rate of inflammation on wound reduced (macrophage& Neutrophil increase) compounds present in gel cause therapid fibroblast transformation. Further administration of gelexperimental seems to increase DNA and tissue power andstimulates the formation of new vessels and epithelization.Application of gel causes the increase in collagen synthesis andthus the healing is performed quicker (Crindly and Reynolds1986; Brown and Mcdonnell 1988; Chithra et al. 1998;Takamiya et al. 2003). On the 7th day, which is considered as theproliferation phase of the healing process, increase in numberof fibroblasts in experimental group 1 is decelerated, but thenumber of fibroblasts in experimental group 2 increases. Theseresults show the capacity which potentially exists in the woundhealing process and twice application of the gel activates thisreaction more. The wound diameter on the 7th day is lower inexperimental group 2 and the wound heals better. The structurerenewal of the wound healing process occurs after the significantacceleration in proliferation phase.

On the 14th day, this is considered as structure renewal ofthe wound healing process, in the experimental groups, excess inthe number of fibroblasts as compared with the control groupand even as compared with day 7, mentions the continuousproliferation phase of healing process till day 14. Significantand simultaneous increase in fibroblasts and decrease in thewound thickness of the experimental groups shows the effect ofgel on increasing the proliferation and fibroblast metabolism.Decrease in number of macrophage in experimental group 2was in agreement with the condition of this day, because of theapplication of gel, the extra gel particles present in the woundmatrix, the number of neutrophils in experimental groups,especially experimental group 2, increases and the number of

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vessels and endothelium in experimental group 2 decreased.Wound diameter in experimental group 2 decreased greatlybecause of twice administration of gel and the power of woundhealing was more than the other groups. Administration of theAloe Vera gel twice daily on dermal wound in wistar rat causessignificant and better results compared to administration oncedaily.

Declaration of interest: The authors report no conflicts ofinterest. The authors alone are responsible for the content andwriting of the paper.

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77 Influence of Aloe Vera Gel on Wound Healing

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