{"id":5849,"date":"2015-12-28T10:06:28","date_gmt":"2015-12-28T10:06:28","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=5849"},"modified":"2020-04-25T06:05:16","modified_gmt":"2020-04-25T06:05:16","slug":"comparison-of-different-concentrations-of-calendula-officinalis-gel-on-cutaneous-wound-healing","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol8no2\/comparison-of-different-concentrations-of-calendula-officinalis-gel-on-cutaneous-wound-healing\/","title":{"rendered":"Comparison of Different Concentrations of Calendula Officinalis Gel on Cutaneous Wound Healing"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Tissue repair and wound healing are\u00a0complex processes that involve inflammation,
\ngranulation, and tissue remodeling. Skin damage\u00a0can occur as a result of burns, cuts, abrasions and\u00a0ulcers to varying degrees of severity. If not repaired,\u00a0any breach of the skin may compromise its barrier\u00a0function and expose the body\u2019s tissues to microbial
\ninfections and mechanical damage. The healing of\u00a0cutaneous wounds requires complex interactions\u00a0between the dermal and epidermal cells, the extracellular matrix (ECM), and the nervous and\u00a0vascular components of the damaged and
\nsurrounding skin (19, 36).\u00a0Calendula officinalis, or pot marigold, is a\u00a0common garden plant belonging to the Compositae\u00a0family. Native to Southern Europe, Calendula grows\u00a0up to 60 cm in height and produces large yellow or\u00a0orange flowers (60). It is used topically as a natural\u00a0anti-inflammatory medicine and for poorly healing
\nwounds and leg ulcers. The dosages cited are 2\u00a0mL of tincture diluted to 250\u2013500 mL with water\u00a0or 2\u20135 g of herb in 100 g of ointment (53). Other\u00a0topical uses include treatment for 1st degree burns\u00a0and scalds, bruises, boils, and rashes (21, 33).
\nCalendula officinalis has many pharmacological\u00a0properties. It is used for the treatment of skin\u00a0disorders, pain and also as a bactericide, antiseptic\u00a0and anti-inflammatory. Butanolic fraction of\u00a0Calendula officinalis possesses a significant free\u00a0radical scavenging and antioxidant activity (7).\u00a0Calendula officinalis flowers were believed to be\u00a0useful in reducing inflammation, wound healing,\u00a0and as an antiseptic, was used to treat various skin\u00a0diseases, ranging from skin ulcerations to eczema.\u00a0Internally Calendula officinalis has been used for\u00a0stomach ulcers and inflammation. The flavonoids,\u00a0found in high amounts in Calendula officinalis, are
\nresponsible for its anti-inflammatory activity;\u00a0triterpene saponins may also be important.\u00a0Calendula officinalis also contains carotenoids(44).<\/p>\n

Material and Methods<\/strong><\/p>\n

Preparation of the gel\u00a0Dried calendula flower tops were used for\u00a0extraction. Calendula flowers (263.7 g) were\u00a0extracted with 1,200 cc ethyl alcohol 70%. For this,
\nthe flowers were placed in an Erlenmeyer flask and\u00a0the alcohol was added. This flask was stoppered\u00a0and sealed and then placed in a dark room at room\u00a0temperature and shaken every day for 1 week. The\u00a0dark liquid was then decanted. A rotary device was\u00a0used for the next step. This device can separate\u00a0solvent faster and work with a vacuum. If the flask\u00a0was left at room temperature to evaporate, it would\u00a0have taken a long time. When the solvent separated,\u00a0the rest of our mixture was ready and was called\u00a0extract. This apparatus works with ice, and water\u00a0always flows in it. Finally, the vacuum increased\u00a0solvent separation. Ten days later, the oily extract of\u00a0calendula flowers was available. To prepare the\u00a0gel, at first the extract was dried with a freeze dryer\u00a0and then the gel base was made. For this, 1 g\u00a0carbopol was added to 95 cc distilled water (5%\u00a0gel), 1 g to 93 cc distilled water (7% gel), and 1 g to\u00a090 cc distilled water (10% gel); after 4 or 5 h, all of\u00a0the carbopol powder dissolved and sodium\u00a0hydroxide was added to make the gel base. Finally,\u00a05 g (5% gel), 7 g (7% gel), and 10 g (10% gel) of\u00a0extract was added to the gel base and used for all\u00a0experiments.\u00a0Experimental animals\u00a0Seventy five white Sprague-Dawley male\u00a0rats weighing between 180 to 220 g were divided\u00a0into three groups that contained 25 rats, Two of them\u00a050 rats for 14 , 21 days and histopathological testing,\u00a0the other one 25 rats for 21 days and biomechanic\u00a0testing (Table 1). Each one was housed individually\u00a0in a separate standard cage and fed with normal\u00a0mouse chow and water ad libitum. Their information\u00a0was written on their cage. Temperature (25\u00b0C) and\u00a0the ratio of daylight hours to non-daylight hours\u00a0(12:12 h ratio of light to dark) were kept constant.\u00a0The rats were divided into five groups as follows:\u00a0group \u00c9 control without any treatment (n=15), group\u00a0\u00c9\u00c9 gel base with placebo treatment (n=15), group\u00a0\u00c9\u00c9\u00c9 5% gel (n=15), group \u00c9V 7% gel (n=15), and\u00a0group V 10% gel (n=15). Care was taken to avoid\u00a0unnecessary stress to the animals throughout the\u00a0experimental period.\u00a0Surgical procedures\u00a0The rats of all groups were anesthetized\u00a0by injection of 0.8 cc ketamine (5%) and 0.2 cc\u00a0xylazine intramuscularly in the hamstring muscles.\u00a0Before making incisions, the dorsal aspect of the\u00a0cervical or cervical to lumbar area was shaved and\u00a0washed with a scrub solution of povidone\u2013iodine.\u00a0Under sterile conditions, a skin incision was made\u00a0in a square shape 2\u00d72 cm in the cervical region for\u00a0histopathological groups, and a rectangular shape\u00a0with a metal ruler from cervical to lumbar region for\u00a0biomechanical groups, the long side of the defect\u00a0was parallel to the vertebral column on both sides\u00a0and the cranial border 1 cm caudal to the axis\u00a0vertebrae. In all groups only the skin was removed.\u00a0The same procedures were performed for both the\u00a0experimental and control groups. The scalpel blade
\nused to create defects. After operation and\u00a0treatment the area was left without a bandage. The\u00a0duration of anesthesia was about 10 min for each\u00a0rat.\u00a0Treatment regimens\u00a0In the control group, after making the\u00a0incision on the cervical and cervical to lumbar\u00a0region, no treatment was applied on the incision\u00a0and the incisions remained intact. Fifteen rats in\u00a0this group were followed 14 and 21 days later. All\u00a0wounds during the 14 and 21 days were gently\u00a0wiped with a sterile spatula without any gel. Daily\u00a0observation was performed and any wound fluid or\u00a0any evidence of infection or other abnormalities\u00a0were noted. The rats from placebo group received\u00a0the gel base in a thin, uniform layer on the wound\u00a0daily for 14 and 21 days. In the treatment groups,\u00a0the rats received 5%, 7%, and 10% calendula\u00a0flower gel for 14 and 21 days.\u00a0Sampling\u00a0After 14 and 21 days post injury, the rats\u00a0were euthanized by IV injection of 30 mg\/kg\u00a0thiopental sodium (nesdonal) via tail vein and\u00a0sampling was done. Samples (2\u00d72cm) for\u00a0histopathological studies were taken from the edges\u00a0of the wound, so that the sample contained both\u00a0the lesion and its periphery including normal skin.\u00a0Samples from the rest of animals (cervical to lumbar\u00a0region) were collected for tensile testing.\u00a0Biomechanical studies\u00a0Skin from five animals in each group were\u00a0dissected for biomechanical tensile testing and\u00a0stored at \u00d720\u00daC prior to testing. They were then\u00a0thawed at room temperature before tensile testing.\u00a0Throughout the testing period, the specimens were\u00a0kept moist in saline soaked gauze. The specimens\u00a0were mounted in a tensile testing machine\u00a0controlled by a personal computer. The proximal\u00a0section was clamped to the skin using a serrated\u00a0interdigitating mechanical clamp. The distal section\u00a0of the specimen was mounted in a clamp, so that a\u00a0constant specimen length of 1.5cm was used in the\u00a0test. Tensile testing to failure was performed by\u00a0applying 10 kg force at a strain rate of 50 mm min\u2022.\u00a0The ultimate strength was recorded by the computer.\u00a0Histopathological studies\u00a0Skin samples were taken from both the\u00a0wound and adjoining normal skin and fixed in 10%\u00a0neutral buffered formalin. After fixation, the tissues\u00a0were embedded in paraffin, and 5\u03bcm thickness\u00a0sections were stained using hematoxylin and eosin.\u00a0Five zones were examined from the sample\u00a0morphometrically through a calibrated ocular on a\u00a0Nikon light microscope (Nikon, Tokyo, Japan) at\u00a0magnification of \u00d740, \u00d7100 and\u00d7400. The criteria\u00a0that were studied in the histopathological sections\u00a0consisted of hemorrhage, mononuclear cell\u00a0infiltration, reepithelialization of the epithelium,\u00a0fibroblast content, present of fibrocytes, collagen\u00a0content and neovascularization.\u00a0Statistical analysis\u00a0One-way analysis of variance and\u00a0Duncan\u2019s multiple range tests were used to
\nevaluate the differences of biomechanical\u00a0parameters between the experimental, placebo and\u00a0control groups. Differences were considered\u00a0significant when p\u00c20.05, using computer software\u00a0SPSS version 11.5 for windows (SPSS, Chicago,\u00a0IL, USA).<\/p>\n

Results<\/strong><\/p>\n

Observations during daily care\u00a0On the day postoperative care all of the\u00a0wounds appeared clean. On the 2nd and 3rd days of\u00a0treatment all the untreated wounds developed a\u00a0moderately purulent-appearing exudate and\u00a0inflammation. The treated wounds had no exudate\u00a0during this time and over the 14 and 21 days of\u00a0follow-up, but a slight inflammation was seen. On\u00a0day 14 the wound size in all groups was reduced,
\nbut it seemed the treated animals had smaller\u00a0wound size. On day 21 post-injury, the wound size\u00a0in the treated lesions was smaller than in the control\u00a0one.
\nBiomechanical properties\u00a0As is shown in Table 2 and figure 1 the\u00a0ultimate tensile strengths of the 7% and 10% gel\u00a0groups were significantly higher than those of the\u00a0untreated group at 21 days post-injury (p<0.05).
\nThis value for the 5% gel group was also higher\u00a0than that of the control and placebo groups;\u00a0however, the difference was not significant. The\u00a0ultimate tensile strength of the 7% gel group was\u00a0significantly greater than the 10% gel group\u00a0respectively (p<0.05). The ultimate strength of the\u00a0control and placebo group was similar and showed\u00a0no significant difference.<\/p>\n

Table 1: Test group arrangements<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
<\/td>\n14 days<\/td>\n21 days<\/td>\n21 days<\/td>\n<\/tr>\n
<\/td>\nHistopathological test<\/td>\nHistopathological test<\/td>\nBiomechanic test<\/td>\n<\/tr>\n
Control<\/td>\n5 rats<\/td>\n5 rats<\/td>\n5 rats<\/td>\n<\/tr>\n
Gel base<\/td>\n5 rats<\/td>\n5 rats<\/td>\n5 rats<\/td>\n<\/tr>\n
5% gel<\/td>\n5 rats<\/td>\n5 rats<\/td>\n5 rats<\/td>\n<\/tr>\n
7% gel<\/td>\n5 rats<\/td>\n5 rats<\/td>\n5 rats<\/td>\n<\/tr>\n
10% gel<\/td>\n5 rats<\/td>\n5 rats<\/td>\n5 rats<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Histopathological findings\u00a0At microscopic level, however, the treated\u00a0tissues at 14 days post-injury were not properly\u00a0organized, but re-epithelialization was complete\u00a0and the epithelium was thicker than the control and\u00a0placebo group. Some of the treated lesions did not\u00a0show any inflammatory cells and in others very few<\/p>\n

macrophages and lymphocytes were present. No\u00a0neutrophils were present at the treated lesions in\u00a0this stage. Many fibroblasts and loops of new\u00a0growing blood vessels were seen in the repaired\u00a0area, while in the untreated animals the tissue was\u00a0more unorganized and many inflammatory cells\u00a0including macrophages and lymphocytes were\u00a0present at the site of injury. Fewer fibroblasts and\u00a0loops of new regenerating blood vessels were seen\u00a0in the sections of the untreated animals. These\u00a0changes were more apparent in the 7% gel group\u00a0and the lesions showed more re-epithelialization
\nand positive changes than the other gel groups.\u00a0The 10% and 5% gel groups showed no significant\u00a0differences to each other, but there were differences\u00a0to the control and placebo groups (Table 3). After\u00a021 days of treatment, the thicknesses of the injured
\ntreated skins decreased and were not significantly\u00a0different from the control and placebo groups. At\u00a0this stage, the treated tissues were more organized,\u00a0the epithelium was thinner, and very few\u00a0inflammatory cells were observed in the lesion. The\u00a0number of the fibroblasts decreased and most of\u00a0them changed to mature organized fibrocytes and\u00a0were aligned along the reparative connective tissue.\u00a0The number of blood vessels decreased but their\u00a0calibre was greater than those of the 14 days postinjuries.\u00a0In the untreated lesions, however, still some\u00a0unorganized areas were present, and the\u00a0macrophages and lymphocytes were observed.\u00a0Fibroblasts were more numerous in comparison to\u00a0those of the treated lesions. The blood vessels were\u00a0more numerous and with a smaller calibre than\u00a0those of the treated ones. At this stage the lesions\u00a0in 7% gel groups showed more positive changes\u00a0than the other gel groups. Actually the rate of healing\u00a0in this group was much faster than the other groups\u00a0(Table 4).<\/p>\n

 <\/p>\n

Table 2: Comparison of ultimate tensile strength of treated lesions with different\u00a0Concentrations of calendula officinalis flower gel with control and placebo groups (p<0.05).<\/strong><\/p>\n\n\n\n\n
Gel<\/td>\nControl<\/td>\nplacebo<\/td>\n5% gel<\/td>\n7% gel<\/td>\n10% gel<\/td>\n<\/tr>\n
Ultimate tensile strength (kg)<\/td>\n0.91\u00b10.047<\/td>\n1.16\u00b10.1<\/td>\n1.21\u00b10.13<\/td>\n2.94\u00b10.31*<\/td>\n1.71\u00b10.02*<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

(*) means, groups which are significantly better.<\/p>\n

Discussion<\/strong><\/p>\n

Wound healing is a dynamic, interactive\u00a0process involving soluble mediators, blood cells,\u00a0extracellular matrix, and parenchymal cells (55).\u00a0Though the healing process takes place by itself\u00a0and does not require much help, various risk factors\u00a0such as infection and delay in healing have been\u00a0mentioned to promote this process (48). Calendula\u00a0officinalis flowers have anti-inflammatory effects (8),\u00a0and so this can promote wound healing. Also,\u00a0several components that affect the healing of burn
\nwounds (5), promote anti-tumor (62) and antiedematous\u00a0activity (67) and result in the treatment\u00a0of venous leg ulcers (12) are isolated from this\u00a0flower.\u00a0Adnan Hasanoglu et al., demonstrated\u00a0that microionized flavonoid fractions can be used\u00a0in the treatment of venous leg ulcers (20). The drugs\u00a0tanus- improving effects on blood vessels, its\u00a0regulation of lymphatic drainage and edema\u00a0reducing effect may partly explain the healing of\u00a0ulcers (1, 18). It has been shown in vitro and in vivo\u00a0that micronized flavonoid fraction reduces the\u00a0activation of the complement system (9). It has been\u00a0thought that this mechanism of action constitutes\u00a0the gel\u2019s anti inflammatory effects and\u00a0consequently, may contribute to the healing of\u00a0ulcers. In a study conducted by Lonchampt et al., in\u00a0vitro and in vivo effects of the drug on oxygen\u00a0radicals were examined and it was proved to have\u00a0a protective effect against active oxygen radicals\u00a0(32). Recently, some studies demonstrated the\u00a0favorable effects of micronized flavonoid fraction\u00a0on microcirculation (20). The importance of regular\u00a0microcirculation in wound healing is well-known.\u00a0Delayed healing of wounds bound up tightly has\u00a0been attributed to the development of ischemia due\u00a0to impaired microcirculation (20).\u00a0In the present study, it was observed that\u00a0the use of calendula officinalis in wounds is\u00a0beneficial, based on measurements and\u00a0histopathological evaluations done at days 14 and\u00a021 (p < 0.05). Its effectiveness in wounds is thought\u00a0to be due to its contributions to the reduction of\u00a0edema and the regulation of microcirculation in the\u00a0environment. Adnan Hasanoglu et al., investigated\u00a0whether systemic or topical usage of flavonoids\u00a0fraction produces better and faster healing of
\ninfected wounds in guinea pigs (20). The fact that it\u00a0produced good results in infected wounds rather\u00a0than in clean wounds has suggested that it might\u00a0also have an antibacterial property, and as\u00a0Calendula officinalis has much flavonoid in its\u00a0structure, all of these properties can lead us to the\u00a0results of this research.<\/p>\n

Many plant species accumulate a wide\u00a0range of triterpenic oleanolic acid (OL) derivatives,\u00a0mainly glycosides (58). There is an array of medical\u00a0products derived from some of these plants, which\u00a0are used for the treatment of various diseases and
\nailments (3). Marigold (Calendula officinalis), family\u00a0Asteraceae, well known for its pharmaceutical and\u00a0cosmetic use, contains two series of OL glycosides,\u00a0i.e. glucosides (derivatives of 3-O-monoglucoside)\u00a0and glucuronides (derivatives of 3-Omonoglucuronide)\u00a0(24, 64). All of these compounds\u00a0have biological activity: glucosides are mainly\u00a0allelopathic and hemolytic agents, and\u00a0glucuronides are potent fungistatics. It has also\u00a0been shown that all glycosides of OL are\u00a0synthesized in leaves (25, 59). So we used flowers\u00a0but no allelopathic or hemolythic effects were seen.<\/p>\n

Table 3: Cells and Vessels numbers in different groups after 14 days (significant difference between groups p<0.05<\/em>).<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
 <\/p>\n

 <\/p>\n

Cells<\/strong><\/p>\n

and Vessels number<\/strong><\/td>\n

Gel groups<\/strong><\/td>\n<\/tr>\n
<\/td>\n5% gel<\/td>\n7% gel<\/td>\n10% gel<\/td>\nGel base<\/td>\nControl<\/td>\n<\/tr>\n
Angiogenesis<\/td>\n7\u00b10.54<\/td>\n8\u00b10.7*<\/td>\n6\u00b10.31<\/td>\n3\u00b10.54<\/td>\n4.8\u00b10.37<\/td>\n<\/tr>\n
Lymphocyte<\/td>\n16\u00b11.78<\/td>\n12.2\u00b11.49*<\/td>\n20.2\u00b12.7<\/td>\n21.4\u00b12.6<\/td>\n22\u00b11.51<\/td>\n<\/tr>\n
Plasma cell<\/td>\n0.8\u00b10.2*<\/td>\n0.8\u00b10.2*<\/td>\n1.6\u00b10.24<\/td>\n1.6\u00b10.24<\/td>\n2\u00b10.00<\/td>\n<\/tr>\n
Macrophage<\/td>\n2.4\u00b10.74*<\/td>\n1.8\u00b10.2*<\/td>\n1.6\u00b10.24<\/td>\n8.4\u00b11.8<\/td>\n8.8\u00b12.35<\/td>\n<\/tr>\n
Fibroblast<\/td>\n40\u00b19.9<\/td>\n60.6\u00b18.69*<\/td>\n24.4\u00b11.66<\/td>\n22.4\u00b13.7<\/td>\n17.6\u00b11.8<\/td>\n<\/tr>\n
Fibrocyte<\/td>\n9.4\u00b10.74<\/td>\n14.2\u00b12.8*<\/td>\n3.4\u00b10.67<\/td>\n2.8\u00b1.37<\/td>\n4\u00b10.31<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

(*) means, groups which are significantly better.<\/p>\n

Nikiema et al., also showed that triterpenoids can\u00a0promote keratinocyte proliferation due to their antiinflammatory\u00a0effects (38).\u00a0This study was designed to determine
\nwhether or not the topical application of different\u00a0concentrations of Calendula officinalis gel in two\u00a0different days is an effective means of prompting\u00a0wound healing. Significant positive effects on\u00a0wound healing were detected by the three different<\/p>\n\n\n\n
\"figure<\/td>\nFigure 1: Comparison of ultimate tensile strength of the treated lesions with different concentrations of calendula officinalis flower gel with the control and placebo groups. (p<0.05)<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"figure<\/td>\nFigure 2: Histopathology section of tissue treated with 5% gel after 14 days. Magnification: 4<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"figure<\/td>\nFigure 3: Histopathology section of tissue treated with 7% gel after 14 days. Magnification: 4<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"figure<\/td>\nFigure 4: Histopathology section of tissue treated with 10% gel after 14 days. Magnification: 4<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"figure<\/td>\nFigure 5: Histopathology section of control group after 14 days. Magnification: 10<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"figure<\/td>\nFigure 6: Histopathology section of tissue treated with gel base after 14 days. Magnification: 4<\/strong><\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

concentrations used. 7% concentration of this gel\u00a0had significant positive effects on wound healing\u00a0on days 14 and 21, and especially on the 14th day.\u00a0This may be because of the covering of the wound\u00a0after 21 days by scar tissue, so if we can use this\u00a0flower flos extract parenterally it will be more\u00a0effective. Walter B. Mors et al., showed that some of\u00a0the components of this flower can be used\u00a0parenterally to neutralize snake venom (37).\u00a0TRAUMEEL is an anti-inflammatory, antiedematous,\u00a0anti-exudative combination formulation\u00a0of 12 botanical substances including calendula\u00a0officinalis and 1 mineral substance. It is officially\u00a0classified as a homeopathic combination remedy\u00a0(56). This flower can be used parenterally for\u00a0homeopathic therapy and the recommended\u00a0maximum parenteral dose for large animals is 10ml\u00a0per animal. In human homeopathy, lower\u00a0concentrations and topical forms should be used,\u00a0as it can be toxic for humans and the studies are\u00a0not enough (11). Polyphenolic-polysaccharide\u00a0compounds in the Asteraceae family including\u00a0Calendula have chemical characterization and\u00a0blood anticoagulant activity (41). All of these\u00a0publications show that this product can, perhaps,\u00a0be used parenterally.<\/p>\n

Most of the other publications focus on the\u00a0topical application of this gel, perhaps because of\u00a0the probability of intoxication and\/or it will be easier\u00a0to use this product topically. Also, sterility and\u00a0solubility are not important when we use a product\u00a0externally, so because of the probability of\u00a0intoxication and to find out which of the\u00a0concentrations may be toxic, three different\u00a0concentrations were used. The 7% gel was more\u00a0effective than the others (Tables 3 and 4).<\/p>\n

In Histopathological studies, lymphocytes\u00a0and plasma cells were more apparent after 14 and\u00a021 days when the 10% gel was used. These cells\u00a0are the responses for allergy or inflammation, and\u00a0so we can conclude that this concentration is irritant\u00a0to cells. Rea et al., showed some of the components\u00a0of the Calendula flower and Calendula flower\u00a0extracts used in cosmetics and personal care\u00a0products were not safe and could irritate the skin\u00a0(53), but nothing was mentioned about the safe\u00a0doses. Yoshikawa et al., also mentioned that some\u00a0of the components have gastroprotective effects,\u00a0but did not name any dose dependent effects (66).\u00a0Fiume et al., indicated that acute toxicity studies in\u00a0rats and mice showed the extract is relatively\u00a0nontoxic (13). Animal tests showed at most minimal\u00a0skin irritation, and no sensitization or phototoxicity.\u00a0Minimal ocular irritation was seen with one\u00a0formulation and no irritation with others. Six\u00a0saponins isolated from Calendula officinalis flowers\u00a0were not mutagenic in an Ames test, and a tea\u00a0derived from this flower was not genotoxic in\u00a0Drosophila melanogaster, the use of Calendula\u00a0officinalis by oral route in humans needs a safety\u00a0evaluation for this route of administration. Available\u00a0data are insufficient to support the safety of\u00a0Calendula officinalis extract by oral route. Lagarto\u00a0et al., have studied the acute and subchronic oral\u00a0toxicities of Calendula officinalis extract in male and\u00a0female Wistar rats (30). They have dissolved a single\u00a0acute Calendula officinalis extract dose of 2000 mg\/\u00a0kg in distilled water and administered it by oral\u00a0gavage for acute toxicity. Subchronic doses of 50,\u00a0250 and 1000 mg\/kg\/day were administered in\u00a0drinking water. The major toxicological endpoints\u00a0examined included animal body weight, water and\u00a0food intake, selected tissue weights, and\u00a0histopathological examinations. In addition, they\u00a0examined blood elements: hematocrit, hemoglobin\u00a0concentration, erythrocyte count, total and\u00a0differential leukocyte count and blood clotting time.\u00a0Blood chemistry: (glucose, total cholesterol, urea,\u00a0total proteins, alkaline phosphatase, alanine\u00a0aminotransferase (ALT) and aspartate\u00a0aminotransferase (AST)) was examined too. In the\u00a0acute study, no mortality and signs of toxicity were\u00a0found. In the subchronic study, several of the blood\u00a0elements were significantly affected in males and\u00a0females after 90 days; hemoglobin, erythrocytes,\u00a0leukocytes and blood clotting time. For blood\u00a0chemistry parameters, ALT, AST and alkaline\u00a0phosphatase were affected.<\/p>\n

Histopathological\u00a0examination of tissues showed slight abnormalities
\nin hepatic parenchyma that were consistent with\u00a0the biochemical variations observed. These studies\u00a0indicate that the acute and subchronic toxicities of\u00a0Calendula Officinalis extract are low. Bashir et al.,\u00a0indicated that the crude extract of Calendula\u00a0officinalis flowers contains both spasmolytic and\u00a0spasmogenic constituents, exhibiting these effects\u00a0through calcium channel blocking and cholinergic\u00a0activities and so provided a scientific base for its\u00a0traditional use in abdominal cramps and\u00a0constipation (2), so perhaps if some of the useful\u00a0components were extracted and worked to produce\u00a0our gel, the results could be different. Flavonoids\u00a0and terpenoids have protective properties; these\u00a0are the two important components of this flower.\u00a0Aqueous and aqueous-ethanol extracts of\u00a0Calendula officinalis have fewer toxic effects (43),\u00a0so because aqueous-ethanol extract was used, this\u00a0flower gel has had fewer toxic effects. Hexaneextracted\u00a0Calendula meal was tested in an\u00a0acceptance trial with eighty 8\u201313 week old\u00a0crossbred pigs to determine their response to diets\u00a0containing 0, 2, 6, 10 or 20% Calendula meal.\u00a0Performance parameters included feed intake, daily\u00a0growth and post mortem histopathological\u00a0examination of vital organs. Pigs fed a diet\u00a0containing 2% calendula meal consumed\u00a0significantly more feed than the ones fed a\u00a0Calendula-free diet. As the Calendula meal content\u00a0of the diet increased, feed intake tended to\u00a0decrease. Post mortem examination of the vital\u00a0organs showed statistically significant (P < 0.01)\u00a0between-treatment differences in heart, kidney,\u00a0thyroid and pancreas size expressed as percentage\u00a0of bodyweight. The results of blood and blood serum
\nanalyses for haematocrit, haemoglobin,\u00a0oxygenated haemoglobin, aspartateaminotransferase,\u00a0alanine-aminotransferase,\u00a0lactate dehydrogenase, creatinine and zinc\u00a0remained within the reference values for young\u00a0pigs.<\/p>\n

Although Calendula meal showed\u00a0potential as a ration ingredient for young pigs, it is
\nnot advised to include more than 10% of it in the\u00a0diet (23).\u00a0Compared with those of the untreated\u00a0animals, the injured area of the treated group\u00a0showed a gross decrease in the wound thickness\u00a0at 14 days post-injury. This was relevant to the\u00a0decrease in the inflammatory cells population at\u00a0the site of the lesions, together with better\u00a0maturation of the reparative cells and improved\u00a0alignment of the fibroblasts, collagen fibers and\u00a0blood vessels. Dovi et al., investigated whether\u00a0neutrophils in diabetic mice are much higher in\u00a0injured tissue than in healthy mice. They concluded\u00a0that more neutrophils in the wound delay healing\u00a0and, as can be seen, this gel can decrease\u00a0neutrophils in the wound due to its anti-inflammatory\u00a0effects, and can accelerate healing (10).<\/p>\n

This supplement has prompted the\u00a0proliferation of the fibroblasts, endothelial cells and\u00a0epidermal cells. With oral administration of\u00a0calendula officinalis slight histological changes\u00a0were observed in the liver, exhibiting increased\u00a0mononuclear cells in portal triads with the\u00a0prevalence of lymphocytes. Activated Kupffer cells,\u00a0inflammatory cells and focal necrosis, lymphocytes\u00a0aggregation and focal hepatocellular atrophy were\u00a0observed in females. Perivascular aggregation of\u00a0lympho-reticular cells in the portal triad was also
\nobserved (30).<\/p>\n

These effects were also seen in the\u00a010% gel group in this study. Histopathological\u00a0results showed more aggregation of lymphocytes\u00a0and plasma cells and decreased in fibroblast cells.\u00a0Hexane and ethanolic extracts of Calendula\u00a0officinalis stimulated the proliferation and migration\u00a0of fibroblasts at low concentrations, e.g. 10 \u00ecg\/ml\u00a0enhanced cell numbers by 64.35% and 70.53%,\u00a0respectively (16). Regarding the mode of action of\u00a0plant polysaccharides as wound-healing\u00a0promoters, the so-called mucilagineous effect; the\u00a0bioadhesion of polysaccharides to epithelia was\u00a0demonstrated for some traditionally used herbs
\nsuch as Calendula officinalis and it was seen that\u00a0the wounds of the treated groups healed\u00a0considerably sooner than the untreated ones (54).\u00a0It may be due to the bioadhesion of\u00a0polysaccharides to epithelia. Mors et al., used in\u00a0vitro scratch assays to examine the relative\u00a0contribution of dermal fibroblasts in the wound\u00a0repair process and they showed that this process\u00a0will accelerate by fibroblast production (37).\u00a0Experiments of Fonseca et al., demonstrated that\u00a0small concentrations of marigold extracts are\u00a0capable of stimulating the proliferation of mouse\u00a0fibroblasts, with an approximately 27% increase of\u00a0viability observed in cells treated with 11.25 and\u00a015 mg\/mL of this extract (14). These results support\u00a0the observations of Matysik et al., who showed that\u00a0marigold extract in small concentrations can\u00a0stimulate the proliferation of human fibroblasts, but\u00a0at high concentrations it can be toxic (35).<\/p>\n

There is almost unanimous agreement\u00a0that collagen performs a major role in restoring\u00a0strength and remodeling scar tissue (34). Though\u00a0the major function of collagen is to provide strength\u00a0and integrity to the wound (63), it also plays a role\u00a0in other functions such as hemeostasis, reepithelialization,\u00a0cell\u2013cell and cell\u2013matrix\u00a0interactions (6, 50). Not only collagen, but its\u00a0degradative products also function in the healing\u00a0process. For example, they are chemotactic to blood
\nmonocytes (45).<\/p>\n

Since inflammatory cells need to cross the\u00a0extracellular matrix during skin repair following UV\u00a0irradiation, it may be that the increase in\u00a0gelatinases, which degrade most matrix\u00a0extracellular molecules, may be beneficial for skin\u00a0healing. Furthermore, the increase in both\u00a0gelatinases (MMP-9 and MMP-2) induced by the\u00a0ME in irradiated skin may be beneficial for\u00a0procollagen synthesis, regulation of the\u00a0inflammatory response and rearrangement of\u00a0damaged skin. However, future studies addressing\u00a0these possibilities are required (14). So the toxic\u00a0effects of high concentrations could be due to the\u00a0exorbitant production of these gelatinases, which
\ncan destroy all pro-collagen fibers in the tissue.\u00a0Radioemulsions containing trolamine were hoped\u00a0to be \u201cradioprotective\u201d because they are\u00a0macrophage cell stimulators that remove necrotic\u00a0tissue, promote fibroblast proliferation, and, ex vivo,\u00a0reduce vascular alterations, promote epithelial cell\u00a0proliferation, and collagen secretion. The oil-inwater\u00a0formulation also softens nonviable tissues. \u00a0Although controlled studies show no clinical\u00a0radioprotective effect over the control agents, many\u00a0patients express satisfaction with these ointments\u00a0and find them soothing. Pommier et al., compared\u00a0the topical application of Calendula officinalis with\u00a0Trolamine for the prevention of acute dermatitis\u00a0during irradiation for breast cancer (44).<\/p>\n

They\u00a0concluded Calendula is highly effective for the\u00a0prevention of acute dermatitis of grade 2 or higher\u00a0and should be proposed for patients undergoing\u00a0postoperative irradiation for breast cancer. We can\u00a0conclude from these two studies that calendula can\u00a0be used to treat radiation dermatitis because of\u00a0prompting fibroblast production and collagen\u00a0secretion. Preethi et al., investigated there was a\u00a0significantly higher amount of hydroxyproline in the\u00a0granuloma tissue excised from the burned skin of
\ndrug treated animals compared to controls on both\u00a0time periods (47). The hexosamine content was\u00a0found to be decreased during thermal burn but was\u00a0significantly higher in drug treated groups on both\u00a0the 5th as well as the 10th day of burning. This could\u00a0be due to increased synthesis or the decreased\u00a0catabolism of collagen due to the presence of\u00a0flavonoids in the extract which can produce artificial
\ncross linkage between collagen molecules. Also,\u00a0there is a significant increase in the glutathione\u00a0content in all animals in the treatment with the\u00a0Calendula extract (47), so this flower can increase\u00a0the antioxidant activity of injured tissues and\u00a0accelerate their healing. The phytochemical\u00a0constituents of Calendula officinalis include\u00a0flavonoids like lupeol, quercetin, protocatechuic\u00a0acid etc. and many alkaloids and triterpinoids (35).<\/p>\n

Flavoxanthin, luteoxanthin, lycopene,\u00a0auroxanthin, lutein, \u00e2-carotene etc. are the major\u00a0carotenoids present in this flower (27). Most of these\u00a0constituents are reported as free radical\u00a0scavengers and enhance wound healing by\u00a0producing artificial cross linkage (29). The\u00a0production of free radicals at or around the wound\u00a0bed may contribute to delays in wound healing\u00a0through the destruction of lipids, proteins, collagen,\u00a0proteoglycan, and hyaluronic acid (65). Agents that\u00a0demonstrate significant antioxidant activity may,\u00a0therefore, preserve viable tissue and facilitate\u00a0wound healing. Given that the butanolic extract of\u00a0Calendula demonstrates free radical scavenging\u00a0activity against superoxide radicals and hydroxyl\u00a0radicals in vitro in a dose dependent manner; that\u00a0the same extract inhibits iron ascorbate-induced\u00a0lipid peroxidation in rat liver microsomes (7), and\u00a0that several organic solvent extracts of Calendula\u00a0inhibit lipid peroxidation of liposomes in vitro (26),
\nit is argued that Calendula may facilitate wound\u00a0healing via an important antioxidant effect. However\u00a0clinical research is needed to validate these\u00a0findings. The most important clinical endpoint in\u00a0wound management is wound closure or 100%\u00a0epithelialization. Given that wound closure is\u00a0critically important, it is argued that any agent\u00a0demonstrating significant wound-healing activity\u00a0should be seriously considered in conventional\u00a0practice. Calendula, for example, may facilitate\u00a0wound healing by increasing both wound\u00a0angiogenesis (17) and collagen, nucleoprotein, and
\nglycoprotein metabolism (4, 28), leading to\u00a0improvements in both local circulation and\u00a0granulation tissue formation (22). Several\u00a0experimental studies lend support to these claims\u00a0demonstrating that the daily application of a 1:10\u00a0alcoholic extract of Calendula or Calendula cream\u00a0to paravertebral incisions in rats facilitates collagen\u00a0maturation and epithelialization within 10 to 25 days\u00a0(31). Preethi et al., investigated that oral and topical\u00a0application of Calendula officinalis flower extract\u00a0affects excision wounds made in rats (46). The\u00a0parameters assessed with them were the days\u00a0needed for re-epithelization and percentage of\u00a0wound closure. The hydroxy proline and\u00a0hexosamine content in the granuloma tissue of the\u00a0wound was also measured. It was found that the\u00a0percentage of wound closure was 90.0% in the\u00a0extract-treated group, whereas the control group\u00a0showed only 51.1% on the eighth day of wounding\u00a0(p <0.01), and also the days needed for reepithelization\u00a0in their research were 17.7 for the\u00a0control animals; extract treatment at a dose of 20 or\u00a0100 mg\/kg b.wt reduced the period to 14 and 13\u00a0days, respectively. They observed a significant\u00a0increase in the hydroxy proline and hexosamine\u00a0content in the extract-treated group compared with\u00a0the untreated animals. This data indicate the potent
\nwound healing activity of Calendula officinalis\u00a0extract. As seen, 20 mg\/kg and 100mg\/kg body\u00a0weight of the extract have similar effects, so this\u00a0research can confirm our findings as well. E.A. Torres\u00a0Vargas et al., used calendula officinalis flower\u00a0extract to produce hyperbranched polyglycerol\u00a0electrospun nanofibers for wound dressing\u00a0applications and observed that this flower can act\u00a0on collagen deposition, and so, can affect wound\u00a0healing (63). To summarize the results of this study,\u00a0C. officinalis gel can affect collagen deposition and\u00a0wound healing, but not all of its concentrations\u00a0because there is both a dual and an opposite effect\u00a0in different concentrations of this gel. Low\u00a0concentrations have no effects and high\u00a0concentrations have cytotoxic effects.\u00a0All these criteria resulted in improved
\nbiomechanical properties of the injured treated\u00a0tissues compared with those of the untreated ones.\u00a0We assume that reduction of the inflammatory cells\u00a0and tissue oedema, in addition to improved\u00a0maturation and alignment of the connective tissue
\nat the site of injury at 21 days post-injury resulted in\u00a0an increase of tissue ultimate strength (40).
\nIncreasing the tissue ultimate strength showed an\u00a0enhancement in the collagen quantity and quality\u00a0at the site of injury of the treated lesions (40). This\u00a0criterion also showed that the inflammatory phase\u00a0or possibly the beginning of the fibroplasia stage in\u00a0the lesions of the treated group was shorter than\u00a0those of the untreated animals. The presence of\u00a0neutrophils and numerous macrophages and\u00a0lymphocytes in the unorganized untreated tissues\u00a014 days post injury showed that this drug may have\u00a0been effective, mostly in the inflammatory phase or\u00a0at the beginning of the fibroplasia.<\/p>\n

Maximum load, which is the functionally\u00a0most important parameter for characterizing healing\u00a0wounds (49), reflects the ultimate tensile strength\u00a0of the specimen, at which complete failure occurs\u00a0rapidly, and load supporting ability of the tissue is\u00a0substantially reduced (39). This happens as the\u00a0intermolecular cross links are broken and the\u00a0collagen fibrils pass each other or as collagen fibrils\u00a0lose contact with basic substance (15). Higher\u00a0ultimate strength of the treated tissues confirmed\u00a0our Histopathological observations regarding more\u00a0fibroblasts, fewer epithelial gaps and increased\u00a0collagen synthesis in the treatment group.\u00a0Calendula officinalis can precipitate wound healing\u00a0by its anti-inflammatory effect. The faster course of\u00a0inflammatory phase can induce the earlier initiation\u00a0of collagen production (61), and this may explain\u00a0the increase of wound tensile strength on the 21th\u00a0day. However, there are also other mechanisms of\u00a0the effect of this herb on wound healing. Antioxidant\u00a0activities can improve the proliferation of cells into\u00a0the injured area and so accelerate the synthesis of\u00a0collagen (57). Other authors report the acceleration\u00a0of the healing process by contraction of the wound\u00a0area and increasing the wound tensile strength (52).\u00a0Rane et al., refers to the increase in the amount of
\nhydroxylproline in granulation tissue in excisional\u00a0wounds, indicating rapid collagen turnover and\u00a0leading to accelerated healing (51).<\/p>\n

From these findings it can be concluded\u00a0that the application of calendula officinalis flower\u00a0gel has negligible positive effects on the early\u00a0stages of wound healing in experimentally induced\u00a0cutaneous wound healing in rats. However, the 7%\u00a0gel resulted in a better tissue alignment, collagen\u00a0fibrils differentiation and maturation, while the 10%\u00a0and 5% gel showed better results to the controland placebo groups, so the 7% gel is more\u00a0effective, especially for the first fourteen days\u00a0because the wound has less cover and this gel has\u00a0fewer toxic effects.<\/p>\n

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