ORIGINAL ARTICLE
The Effect of Saffron (Crocus Sativus) Extract for Healing ofSecond-degree Burn Wounds in Rats
Ghasemali Khorasani,1 Seyed Jalal Hosseinimehr,2, 3 Peyman Zamani,2Maryam Ghasemi4 and Amirhossein Ahmadi2
2

1
Department of Surgery, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
3
Pharmaceutical Research Center, Mazandaran University of Medical Sciences, Sari, Iran4
Department of Pathology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran(Received for publication on December 23, 2007)(Revised for publication on March 13, 2008)(Accepted for publication on April 17, 2008)

Abstract. The aim of this study was to evaluate the efficacy of pollen of saffron extract
cream in the treatment of thermal induced burn wounds and to compare its results with silver sulfadiazine (SSD) in rats. Animals were divided into four groups and administrated a
topical cream including control, base, saffron (20%) or SSD (1%) at 24 hour after a burn injury that was induced by hot water. In special days, according to a pre-planned schedule, animal’s weight, wound size, as well as skin histo-pathology were determined in different
groups under topical treatments. On day 25, average size of wound was 5.5, 4, 0.9 and 4.1
cm2 in control, base, saffron and silver groups. The wound size of saffron group was significantly smaller than other groups. Histological comparison has shown that saffron significantly increased re-epithelialization in burn wounds, as compared to other cream-treatedwounds. Although the exact mechanism of saffron is unclear, anti-inflammatory and antioxidant effects of saffron may have contributed to the wound healing. The results of this study
raise the possibility of potential efficacy of saffron in accelerating wound healing in burn injuries. (Keio J Med 57 (4) : 190－195, December 2008)
Key words: burn, saffron, wound, silver sulfadiazine

Introduction
Thermal burn injury is a major cause of death and disability, with high cost in health care. Healing of burnwounds still remains a challenge to modern medicine,
though many antiseptics have been discovered. Burnmanagement entails significant duration of hospital stay,
expensive medication, multiple operative procedures andprolonged period of rehabilitation. This makes burn carean expensive proposition and every effort should bemade to provide a shorter in-patient care for the burn patients.1 Recently we showed that a significant number ofpatients exposed to thermal and boiling burn injury inNorthern Iran, in which they stayed for a long time in thehospital for the treatment. More than sixty percent (60%)

stayed between 8 to 30 days.2 Silver sulfadiazine (SSD)
cream at 1% is the most widely used topical treatment
for burn injury. The anti-microbial efficacy of SSD is
probably the main reason why the use of this agent is so
widespread.3 Delayed wound healing following treatment is the most important clinical adverse effect of silver topical agent. The wound healing effects of silver
sulfadiazine was compared with vaselinated tulle gras inburned patients. It was demonstrated that silver sulfadiazine delayed wound healing.4 Several adverse reactions
and side effects have been reported, such as resistance tosilver sulfadiazine, renal toxicity, and leukopenia; therefore confirming that this topical cream should not be
used for long periods on extensive wounds.4 Sloughingof dead tissue in partial thickness burns is retarded be-

Address for correspondence: Faculty of Pharmacy, 18 Kilometer Khazar Boulevard, Mazandaran University of Medical Sciences, Sari, Iran Phone:+98-151-3261244, Fax: +98-151-3261244, E-mail: sjhosseinim@yahoo.com, sjhosseinim@mazums.ac.ir
190

Keio J Med 2008; 57 (4): 190－195

cause silver sulfadiazine delays or prevents colonization
by micro-organisms, and silver sulfadiazine might also
slow down the healing of the wound.5 Prolonged application of partial-thickness burn wounds results in highpatient cares cost and complicates wound healing whilea patient is being followed in the hospital. It is importantto find more effective drugs with shorter time application.
Crocus Sativus (family: Iridaceae) is a herb with a long
history of spicy food uses in many countries. The stigmas of this herb are used as a delicious additive on
foods. This herb was assessed to have many pharmacological properties such as anti-inflammatory, antioxidant,
anti-tumor and anti-depressant effects.6-9
The stigmas of Crocus sativus are expensive and exported mainly from Iran to other countries in the world.
The pollen of stamens is an excessive part of Crocus sativus that is not used as a food additive and is very inexpensive. Fallah Hosseini et al, showed extract of pollenof this herb has a theraputic effect on wounds induced bymustard in animals. It was more effective than control
group.10 Realizing the potential use of pollen from thisherb in wound healing, the present study was undertaken
to study the effect of pollen extract of Crocus Sativus, incomparison with silver sulfadiazine on the rate of burnwound healing.
Materials and Methods
Preparation of Crocus Sativus extract
The pollen of Crocus Sativus was collected from Saraban in the Khorasan province of Eastern Iran. This province is the main producer of saffron in the world. The
pollens of the saffron are dried at room temperature andpowdered in a grinder. Aqueous ethanol (75%) was added to the powdered peels (500 g), and stirred for one
hour. The mixture was kept at room temperature for 48
hours. Following filtration, ethanol was evaporated under reduced pressure at 40ºC. The remained water extract
was dried at oven temperature of 50ºC. As a result, 150
grams of extract powder were obtained.
Preparation and formulation of saffron cream
Two grams of liquid white paraffin, 7.5 grams of sterile alcohol, 3 grams of solid white paraffin, and propylene paraben (0.015 g) were mixed and heated to a boiling point as aqueous phase. Twenty grams of pollen saffron with 70 mL deionized water were added to the mixture of 7 grams of propylene glycol, 3 grams of sodium
luryl sulfate and 0.025 grams methyl paraben. The mixture was heated as organic phase. Then, two separate
phases were mixed continuously while being treated to acooler temperature. Thus, the uniform cream was pro-191

duced after cooling the cream filled in tube. Cream contained 20% of pollen. Our experimental research andformulations were carried out under sterile conditions.
The final creams were tested for any probable contamination microbes.
Test animals
Male Wistar rats (n = 48) weighing 180 to 200 grams
were used and housed under standard conditions at room
temperature and given laboratory food and water, ad libitum throughout the study. The experimental protocol wasapproved by Research Committee of Mazandaran University of Medical Sciences prior to commencement of
this study. The weights of the animals were measured.
They were anesthetized intraperitoneally with thiopental(40 mg/kg body weight), while their backs were shaved.
They were then placed in a supine position inside thebottom of hot water (90ºC for 6 seconds). This heat exposure caused a uniform second-degree burn on the backof the skin. In this experiment, approximately 10% total
body surface (TBSA) was burned. The animals were resuscitated with an intraperitoneal injection of 5 ml ofnormal saline solution.
After 24 hours, the animals were assigned at random tofour groups (n = 12/group). Group 1 was the control
group and no topical agent was applied, group 2 wasbase cream, (treated group only with base cream without
effective agent), group 3 was 1% SSD (silver sulfadiazine, Behvarzan Pharmaceutical Company, Rasht, Iran),group 4 was with a 20% saffron cream treated group.
Treatment began 24 hours after the burn injury. Wounds
were treated twice daily.
In order to quantify the rate of wound healing, the sizeof lesions was determined at 1, 3, 7, 10, 14, 20 and 25days after burn injury be well apparent. At this time,each test animal was held in a good position and woundmargin was traced on a transparent plastic sheet using afine-tipped pen. Lesion body area was displayed as cm2
on each experiment day. The area of the wounds on thefirst day was considered as 100% and wound areas on
subsequent days were compared with the wound area on
the initial days. The animals were weighed on the different days after the burn injury.
Histological study
Due to the wound healing of burn wounds, re-epithelialization was evaluated after 25 days and the last day oftreatment period. For this purpose, skin tissue samples
were taken for histological studies with a small excisioncontaining part of the wound area. Tissues were fixed in
10% formalin. Paraffin-embedded sections (5-μm thick)were prepared and stained with hematoxylin and eosin.
Light microscopy was used to evaluate the pathologicalKhorasani G, et al: The effect of Saffron on Wound Healing

192

Table 1 Body weights of rats treated with base, saffron and silver sulfadiazine creams and control during the period of these experiments.
GroupsControl (g)Base (g)Saffron (g)Silver sulfadiazine (g)1
176±17180±20177±18183±124
170±18180±18170±15178±10Days after burn injury
710
14
173±18183±17189±19184±13
189±14
189±11
168±11169±15175±16176±11177±15185±1120200±22203±18189±19192±1325
208±24206±16191±16190±12

Table 2 Average size of wound in animals as control and treated with base, saffron and silver sulfadiazine creams.
Groups*Control (cm2)
Base (cm2)
Saffron (cm2)
Silver sulfadiazine (cm2)728.9±2.928.4±8.3
30.1±6.5
34±4.4Days after burn injury
101420
26.7±5.621±8
7.5±4
25.3±612.3±5.6
6.8±2.2
24.3±7
16±7
4±1.7
28±6
18.6±8.5
6.6±3.7

25
5.5±3
4±2.3
0.9±1.9**
4.1±3.6* Values are means ± SD for each group of ten mice.
** P<0.05 comparison of saffron extract cream compare to other groups.

changes, e.g., granulation tissue formation and re-epithelialization in wounds and their comparison with the normal tissue part. The severity of inflammation in healed
areas was evaluated by counting the mean number of inflammatory cells infiltration per high field (PHF) (×400
magnification) of three samples of each groups.
Statistical analysis
One-way analysis of variance (ANOVA) and tukey
post test were used to compare group means. The differences were considered significant, when p<0.05.
Results
Average weight of animals in four groups of control,base, saffron and silver sulfadiazine before beginning of
this study was 176, 180, 177 and 183 grams, and after 4days of burning was decreased to 170, 180, 170 and 178
grams respectively. There were no significant differences
between groups in the reduction of weight. After thetreatment period (25 days), average weight of animal increased (Table 1).
Wound condition on the 7th day after the burn, was assessed macroscopically in all groups for facilitation of
exact determination of wounds size. Skin lesions were
measured on 7, 14, 20 and 25 days after burn injury andits ratio to first day of burn was evaluated. Average sizeof wound after 25 days was 5.5, 4, 0.9 and 4.1 cm2 incontrol, base, saffron and silver groups (Table 2). There

were significant differences between saffron group andthe other three groups in this aspect (p<0.05). There wasno observed statistical difference among control, baseand silver sulfadiazine in wound size on the 25th day oftreatment. Trends of wound size ratios were showed in
the Figure 1 in control and treatment groups. The photograph of burned mice on the 4th and 10th of treated withsaffron cream is demonstrated in Figure 2.
Results of the histological studies indicated an improvement in wound healing on the 25th day after treatment. The slides from saffron treated wounds were
showed significant wound healing with completed re-epithelialization of epidermis, fibrosis of dermis and mild
inflammatory cell infiltration. (Fig 3). The severity of inflammation in healed areas was approximately 12, 20
and 28 inflammatory cell infiltration for saffron, silver
and base cream treated groups respectively. The slides ofwounds treated with SSD showed lesser degree of woundhealing compared to saffron and basic creams treatedgroups. Bacteria were found in the wound treated withbasic creams. Histological samples of non-treated control group showed negligible wound healing and many
bacteria.
Discussion
Saffron has a food and medicinal history usage in theworld and is widely used as a food additive. Saffron is
the most precious and expensive spice in the world. The
saffron filaments, or threads, are actually the dried stig-Keio J Med 2008; 57 (4): 190－195

193

Fig. 1 Comparison of wound burn size in control, base, saffron and silver sulfadiazine. The wound size was stated as 100% on the 7th
day and wound size on other days calculated compare to the 7th day.

Fig. 2 Photograph of burned mice on the 4th (left) and 10th (right) of treated with saffron cream.

mas of the saffron flower and have red colors. These
components are often dried and used in cooking as a seasoning and coloring agent and it is most expensive.
There are several reports showing that saffron has pharmacological effects including anti tumor, antidepressant,neuroprotective and antioxidative effects.6, 7, 9, 11, 1 2 The
stamens parts of the flower do not have any taste and areless expensive and yellow in color. Stamens of saffron
flower were used in this study. Assessment of woundsize and microscopical examination were employed forevaluating efficacy of pollen saffron in the treatment ofburn injuries. Our results indicated that saffron cream isable to accelerate the rate of wound healing and shortens
the time of healing, compared with those treated withsilver sulfadiazine in burned rats. The extent of healing
with saffron was significantly higher than silver sulfadiazine and base creams. In our study, microscopic examination of the histo-pathological slides, prepared after thecompletion of treatment, revealed that the wounds treated with saffron cream contained fewer inflammatory
cells than other groups. Wound closure was complete
macroscopically, and the histological studies of woundsection confirmed the formation of epiderm and less inflammatory cells infiltrated the dermis. After treatment
with 20% saffron cream, the epidermis was close to normal structure.
Chemical constituents of saffron include crocins, crocetin, picrocrocin, β-carotene and safranal.9, 1 2 These

194

Khorasani G, et al: The effect of Saffron on Wound Healing

Fig. 3 Macroscopic view and photomicrograph of skin wound section of rat on the 25th day of treated with saffron. Left pathologic
slide is ulcer and healing junction, right pathologic slide is healing skin. Most part of skin was healing with growing hair on skin. The
specimens stained with Hematoxylin and Eosin.

compounds have antioxidant activity.6, 9, 12 Saffron inhibited the growth of tumors in brain of mice.1 3 Ethanolic
extracts of Crocus sativus showed anti-inflammatory effects in animal model,8 Crocetin modified the inflammatory markers such as tumor necrosis factor (TNF) and interlukin (IL).1 4 The release of pro-inflammatory mediators such as IL and TNF and reactive oxygen species are
increased in patients with burn injuries.1 5 Thus, saffron
may affect the healing of burns with its antioxidant andanti-inflammatory effects.
Silver sulfadiazine cream is the most widely used topical treatment for burn injury and the antimicrobial efficacy of SSD is probably the main reason for the usage ofthis agent.7 Although SSD is considered a gold standard
for treatment of burn injuries, prolonged application ofthis agent may result in a longer hospitalization.
We found that the severity of inflammation in SSD
group was more than that in saffron and also base cream.
The inflammatory effect of SSD may lead to a delay inre-epitalization of epidermis and dermal fibrosis. Bothfibroblasts and keratinocytes are susceptible to lethal
damage when exposed to concentration of silver which
are lethal for bacteria. In other words, silver-based products can not discriminate healthy cells involved in woundhealing and bacteria.16
As in our experimental study, delayed wound healing
is often observed clinically following the use of silvercontaining topical antimicrobial agents.1 7 Based on the
result of this study, it is probable that saffron cream
could lead to a shorter hospitalization of burned patientscompared with SSD.
We showed that saffron has better wound healing effects in burn animals compared to SSD. For instance, atthe 25th day of our study, the wound size in saffron group
was 0.9 cm2 compared to 4.5 cm2 in SSD group. In fact,
the re-epithelialization process in animals treated withsaffron was better than those that received SSD. Although the antioxidant and anti-inflammatory effects ofsaffron were suggested, further studies are needed to elucidate the exact mechanism of saffron in wound healing.
Acknowledgment
This work was supported by a grant from Mazandaran
University of Medical Sciences, Sari, Iran. This research
was a student thesis Peyman Zamani for receiving MD
degree in faculty of Medicine, Mazandaran University of
Medical Sciences, Sari, Iran.

Keio J Med 2008; 57 (4): 190－195

References
1. Gore MA, Akholekar D: Evaluation of banana leaf dressing forpartial -thickness burn wounds. Burns 2003; 29: 487－792
2. Khorasani G, Hosseinimehr SJ, Eghbalian A: Epidemiology andmortality of burns in the north of Iran. Res J Biol Sci, 2007; 2:519－522
3. Vloemans AFPM, Soesman AM, Suijker M, Kreis RW, Middelkoop E: A randomized clinical trial comparing a hydrocolloid-derived dressing and glycerol preserved allograft skin in the management of partial thickness burn. Burns 2003; 29: 702－710
4. Atiyeh BS, Costagliola M, Hayek SN, Dibo S: Effect of silver onburn wound infection control and healing: review of the literature.
Burns 2007; 33: 139－148
5. Klasen HJ: A historical review of the use of silver in the treatmentof burns. II. Renewed interest for silver. Burns 2000; 26: 131－138
6. Keyhani E, Ghamsari I, Keyhani J, Hadizadeh M: Antioxidant enzymes during hypoxia-anoxia signaling events in Crocus sativus
L. corm. Ann N Y Acad Sci, 2006; 1091: 65－75
7. Akhondzadeh Basti A, Moshiri E, Noorbala AA, Jamshod AH,
Abbasi SH, Akhondzadeh S: Comparison of petal of Crocus sativus L. and fluoxetine in the treatment of depressed outpatients: A
pilot double-blind randomized trial. Progress in Neuro-Psychopharmacol Biol Psychiatry 2007; 31: 439－442
8. Hosseinizadeh H, Younesi HM: Antiociceptive and anti-inflammatory effects of Crocus sativus L. stigma and petal extracts inmice. BMC Pharmacology 2002; 2:7
9. Assimopoulou AN, Sinakos Z, Papageorgiou VP: Radical scav-

195
enging activity of Crocus sativus L. extract and its bioactive constituents. Phytoth Res, 2005; 19: 997－1000
10. Fallah Hosseini H, Kavoli Haghighi M, Jamshidi AH, Pourheidry
GR, Ahmadi AA: The effect of silver sulfadiazine, bee pollen andAloe vera creams against skin side effects induced by thiol mustard. Med Plants 2002; 2: 36－29 (in Persian)
11. Kanakis CD, Tarantilis PA, Tajmir-Riahi HA, Polissiou MG: Crocetin, dimethylcrocetin and safranal bind human serum albumin:stability and antioxidative properties. J Agric Food Chemistry2007; 7: 970－977
12. Premkumar K, Thirunavukkarasu C, Abraham SK, Santhiya ST,
Ramesh A: Protective effect of saffron (Crocus sativus L.) aqueous extract against genetic damage induced by anti-tumor agentsin mice. Hum Exp Toxicol 2006; 25: 79－84
13. Ochiai T, Shimeno H, Mishima KI, Iwasaki K, Fujiwara M, Tanaka H, Shoyama Y, Toda A, Eyanagi R, Soeda S: Protective effectsof carotenoids from saffron on neuronal injury in vitro and in vivo.
Biochem Biophys Acta 2007; 1770: 578－584
14. Yang R, Tan X, Thomas AM, Shen J, Qureshi N, Morrison DC,
Van Way CW: Crocetin inhibits mRNA expression for tumor necrosis factor-alpha, interleukin-1beta, and inducible nitric oxide
synthase in hemorrhagic shock JPEN J Parenter Enteral Nutr.
2006; 30: 297－301
15. Cakir B, Yegen BC: Systemic responses to burn injury. Turk J
Med Sci, 2004; 34: 215－226
16. Poon VK, Burd A: In vitro cytotoxity of silver: implication forclinical wound care. Burns 2004; 30: 140－147
17. Mooney EK: Silver dressings [safety and efficacy reports]. Plast
Reconstr Surg 2006; 117: 666－669