A Prospective, Descriptive Study to Assess the Clinical Benefits of Using Calendula officinalis Hydroglycolic Extract for the Topical Treatment of Diabetic Foot Ulcers

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Ostomy Wound Management 2016;62(3):8–24
Marcelo Buzzi, PhD; Franciele de Freitas, NP; and Marcos Winter, MD

Abstract

Diabetic foot ulcers (DFUs) have a significant impact on patient quality of life. A prospective, descriptive pilot study was conducted between May 2012 and December 2013 through the dermatology outpatient unit in a Brazilian hospital to evaluate the clinical benefits of using Calendula officinalis hydroglycolic extract in the treatment of DFUs.

Patients diagnosed with a stable neuropathic ulcer of  >3 months’ duration; ranging in size from 0.5–40 cm2; without osteomyelitis, gangrene, bone exposure, cancer, or deep tissue infection; ages 18–90 years; with adequate glycemic control and no history of an allergy to C. officinalis were enrolled. Patients provided demographic and diabetes-related information and were evaluated biweekly for 30 weeks or until healing (ie, full epithelialization with no wound drainage). DFUs were measured and clinically examined for microbiological flora and presence of odor, tissue type (eg, granulation, fibrin sloth, necrosis), exudate, and retraction rate using planimetry images. Patients’ blood tests and neuropathic pain assessment (the latter by clinician-directed questionnaire) were performed at baseline and the end of treatment; pain also was assessed during dressing changes using a 10-point rating scale. Patients’ ulcers were treated twice daily with C. officinalis hydroglycolic extract spray solution and covered with saline-moistened, sterile, nonadherent gauze and bandages followed by foot offloading with adequate protective footwear. Patients received their first treatment in the clinic then performed care at home. From a potential population of 109 patients, 25 did not meet the inclusion criteria. Of the remaining 84 participants enrolled, 43 withdrew before study completion; cited reasons included lost to follow-up (16), medical judgment (2), failure to attend >3 scheduled visits (17), protocol violation (5), and death (3). Forty-one (41) — 17 women, average age 62 years (range 44–82 years), average glycemic level 153 mg/dL (range 82–395 mg/dL), most (34) with Wagner type 1 ulcers — completed the study. The proportions of patients who achieved complete wound closure after 11, 20, and 30 weeks of treatment was 54%, 68%, and 78%, respectively; mean healing time was 15.5 ± 6.7 weeks. When individual weekly healing rates (the percentage reduction in wound area per week) were corrected for variability in initial DFU area, the values were nearly 6-fold higher for complete wound closure (7.8% ± 3.6%) than for incomplete wound closure (1.4% ± 0.7%) (Student t-test; P = 0.001). After 30 weeks of treatment, the number of colonized wounds decreased from 29 at baseline to 5, and the number of odorous wounds decreased from 19 to 1. Ulcer bed planimetry data showed a significant reduction in the amount of exudate, fibrin slough, and necrotic tissue after the treatment with C. officinalis hydroglycolic extract (χ2 test; P = 0.001). No adverse events were observed during treatment. The study findings suggest C. officinalis extract is safe and has a beneficial effect on DFU healing. Randomized, controlled studies using C. officinalis hydroglycolic extract are warranted to confirm its safety and establish its clinical efficacy and effectiveness for the topical treatment of DFUs.

 

Diabetic foot syndrome is a common and severe complication worldwide with a cumulative lifetime incidence of up to 25%. The rapidly increasing rates of diabetes make diabetic foot ulcers (DFUs) a major public health issue. These ulcers have been shown to reduce patient quality of life and may ultimately lead to severe pain, prolonged hospitalization, and/or amputation of the lower extremities.1 

Chronic DFUs are complicated by delayed wound healing processes related to impaired glucose metabolism and neurovascular complications.2 The standard treatment plan for DFUs is designed to eliminate infection; maintain a moist wound bed; and offload pressure with protective footwear such as custom cushioned shoes, diabetic boots, and forefoot- and heel-relief shoes, as well as orthotic walkers, wheelchairs, and crutches. Periodic debridement to facilitate healing and topical medication such as preparations made of recombinant human platelet-derived growth factor are also important.3 According to a meta-analysis,4 many patients with persistent DFUs do not respond to commonly provided care. In such patients, a prospective, randomized controlled study5 reports the only available option to prevent amputation is skin replacement therapies. Because skin replacement therapy is expensive and not widely available, more effective treatments for chronic DFUs are desperately needed for patients with diabetes. 

A systematic review6 that included 60 studies (N = 24,747 patients) suggested topical medications and dressings such as cadexomer iodine, zinc oxide, hydrocolloids, alginates, and hydrogels provide no additional benefit to interventions used to treat DFUs such as sharp debridement, larvae therapy, hyperbaric oxygen, skin grafts, electrical and magnetic stimulations, and ultrasound. However, most topical pharmacological agents that are currently available or in development generally address a single aspect of DFU pathology. Examples of research include a randomized, double-blind, placebo-controlled (RDBPC) study7 (N = 87) of abnormal coagulation involving dalteparin, a case series8 (N = 21) using homologous platelet gel, a RDBPC study9 (N = 62) in which infection was treated using a photo-activated gel containing the antimicrobial agent RLP068, and a RDBPC study10 (N = 40) among patients with xerosis treated with a urea-lactic acid moisturizer. 

An emerging trend involves identifying natural extracts with healing properties that address all aspects of DFUs, especially with the new extraction methods designed to optimize their yield, purity, and bioactivity (eg, anti-inflammatory, wound healing, and antitumor properties).11–15 A prospective, randomized controlled trial16 with 37 patients with diabetes demonstrated topical kiwifruit extract reduced the size of DFUs by ~50% in 3 weeks compared to patients who received the standard treatment of surgical debridement, blood sugar control, and oral antibiotic therapy. Additionally, in a prospective, randomized controlled study,17 oak bark extract (QRB7) was found to be more efficient than polyherbal silver sulfadiazine cream for reducing the size of DFUs in 40 patients with diabetes, with 72.5% and 54.7% reduction in wound size after a 6-week treatment. However, none of these agents was proven to support complete wound closure (the duration of the study was not long enough).

The genus Calendula has been recognized for decades as a rich source of medicinal plants with strong healing potencies.16 Among them, Calendula officinalis flowers are used in numerous over-the-counter botanical preparations for external use as an anti-inflammatory wound healing agent and indicated for the treatment of herpes, solar erythemas, burns, and dermatitis. The phytopreparation Plenusdermax® (Phytoplenus Bioativos S.A., Pinhais, Paraná, Brazil) from C. officinalis is rich in various bioactive compounds with wound healing and anti-inflammatory properties, including terpene alcohols, monoester triterpenoids (ie, faradiol and arnidiol calenduladiol), and antioxidant flavonoids (ie, quercetin, rutin, kaempferol, and narcissine). According to animal studies,18–22 the monoesters triterpenoids (ie, isopropyl myristate, palmitate, and laurate) are considered the most effective topical agents in terms of anti-inflammation and wound healing capacity. However, the effects of hydroglycolic extract have not been tested on chronic diabetic ulcers.

 

Study Purpose

A prospective, descriptive clinical study was conducted to evaluate the effect of C. officinalis hydroglycolic extract on the healing rate of DFUs in patients with diabetes until complete wound closure and during a 30-week follow-up period to monitor the long-term effects of the treatment.

 

Methods and Procedures

Participants. The study was conducted between May 2012 and December 2013 in the outpatient unit of the Department of Dermatology, Hospital da Santa Casa de Misericórdia de Curitiba, Pontifícia Universidade Católica do Paraná (PUC-PR), Brazil. One hundred, nine (109) patients with diabetes and foot ulcers from the Curitiba metropolitan region were screened according to the inclusion and exclusion criteria presented in Table 1. The study was approved by the Institutional Research Ethics Committee of the PUC-PR (protocol no. 22.670) and was registered in Plataforma Brazil with the National Commission of Ethics in Research (no. 0.1051212.0.0000.0020). Written informed consent was obtained from all patients before screening. owm_0316_buzzi_table1

At the initial visit, a full medical history and assessment of the patients’ present condition were recorded. Study variables were collected on a paper/pencil instrument and evaluated; information included sociodemographic data, clinical history, and clinical evaluation of the wounds and amputations. The diabetic status of patients (ie, type, duration, glycemic management, including the determination of plasma glucose and glycated hemoglobin levels, current activity level, nutritional status, neuropathic pain assessment,23 and blood tests) also was recorded. Blood test results included glucose levels, glycated hemoglobin, serum albumin, blood count, erythrocyte sedimentation rate, and ulcer bacterioscopy and culture were obtained at baseline and at the study conclusion.

Treatment with C. officinalis hydroglycolic extract. All enrolled patients were treated with the C. officinalis hydroglycolic 4% extract. The extract composition included C. officinalis L. 4% and excipients (butylated hydroxytoluene, parabens, ethanol, polyethylene glycol, and purified water). The spray solution was prepared by authorized compounding pharmacies using commercial ingredients. The certificated analysis of the C. officinalis hydroglycolic extract included the following compounds from dichloromethane fraction: β-amyrin (6.7%), lupeol (4.7%), ψ-taraxasterol (8.1%), calenduladiol monoesters (5.5%), arnidiol monoesters (15.7%), faradiol monoesters (35.2%), others (24.1%), and from aqueous fraction: total flavonoids content of 120 mg/mL. Bioactivity was previously demonstrated using high-performance liquid chromatography.19,20 The DFU was cleaned twice daily with 25 mL of sterile physiological saline solution, after which 0.018 mL/cm2 of wound area of C. officinalis extract was sprayed on the wound. After allowing the solution to dry in the wound bed for 5 minutes, sterile, nonadherent, saline-moistened gauze and bandages were applied. Patients were provided cushioned footwear, diabetic boots, crutches, and wheelchairs to offload the affected areas. None of the patients used additional wound healing medication, phytopreparation, hydrogels, hydrocolloids, or supportive therapy (ie, electrotherapy, vacuum therapy, laser therapy, phototherapy). The nursing team instructed patients or their caregivers to use sterile gauze dressings after each C. officinalis extract application and to avoid bearing weight on the affected limb by using adequate footwear. Patients received their first treatment in clinic then performed care at home. The nursing team monitored whether the instructions for treatment were adequately followed through weekly phone calls to patients or their caregivers. Footwear such as cushioned shoes and diabetic boots was distributed by the local public health system and customized for the study patients. 

Assessment of C. officinalis hydroglycolic extract on DFU healing. DFUs were assessed at baseline and then twice a week during visits by the nursing staff. At each visit, the DFUs were clinically assessed for appearance, size, and size reduction rate using photographs analyzed by computerized planimetry, according to a methodology described previously.24 Photographs of each ulcer were taken with a Sony DSC-H1 digital camera (Sony USA, New York, NY, USA) at every nurse visit. Image capture was standardized using a tripod frame to support the camera fixed perpendicularly to the ulcer. A circular self-adhesive label with a known area was placed close to the ulcer as a calibrator used by the software for area quantification. Digital images obtained were analyzed using Image J® software (National Institutes of Health, Bethesda, MD, USA). The software delineates the margin of each ulcer and uses different shades of color to define and calculate areas with different types of tissue.

The clinical appearance of the ulcers was assessed for different tissue types such as granulation, epithelialization, fibrin slough, and necrosis. The presence of a specific tissue type was confirmed if computerized planimetry showed a relative area >20% of the total ulcer area. The presence of exudate was confirmed by the appearance of moist gauze during dressing changes. The presence of odor was noted. The ulcer was classified according to the Wagner Grading System,25 which was used to establish DFU depth and presence of infection. Microbiological flora of the DFUs was identified using a biogram/antibiogram of a swab from the wound bed. A validated quantitative swab technique was performed to assess wound contamination and infection.26 A wound was considered infected if a high level of bacteria (1 × 106 CFU) and signs of increased erythema, exudate, odor, warmth, edema, and/or pain were present. Patients with infected wounds were treated with systemic antimicrobials; patients who had a fever and other complications from wound infections during the treatment were discontinued during the study.

Complete wound closure (ie, healing) was defined as full epithelialization of the ulcer with the absence of drainage. Patients were monitored biweekly by the study physician and trained nursing staff for 30 weeks or until healing. Healing was confirmed 1 week following closure, and the patient was monitored for another 2 weeks.

If a patient experienced an allergic reaction to the C. officinalis hydroglycolic extract, an evaluation by the principal investigator was conducted, and the patient was removed from the clinical test. 

Pain assessment. Pain was assessed in 2 ways. Patients were asked to complete the paper/pencil Neuropathic Pain Scale Questionnaire (NPSQ) on the first visit (baseline) and after completion of the 30-week treatment under the guidance of the nursing staff for evaluation and discrimination of neuropathic pain, according to the methodology described previously.23 Briefly, patients were rated for numbness (no numbness sensation = 0, worst numbness imaginable = 100), tingling pain (no tingling pain = 0, worst tingling pain imaginable = 100), and increased pain due to touch (no increase at all = 0, greatest increase imaginable = 100). The NPSQ scores for these symptoms were multiplied by its specific coefficients, summed, and then subtracted by a constant to obtain the discriminant function score (DFS). DFS <0 predicted no neuropathic pain, whereas DFS ≥0 indicated neuropathic pain.

For pain related to DFU dressing changes, patients were asked to rate their pain using a numerical rating scale (NRS) that ranged from no pain (0) to the worst possible pain (10).27 The NRS questionnaire was completed at baseline and then once a month during the nursing visits.

Statistical analysis. Descriptive and analytical data were collected and stored using a Microsoft Office Access 2010 database, drawn from documents used in medical evaluations and nursing. Data were analyzed by importing Access files to the database to the Statistical Package for Social Sciences (SPSS) version 20 (SPSS, Inc, Chicago, IL, USA). All data were verified by double key entry as entered and stored in the Access database. The Access database was stored in a computer with an Intel Core 2 Duo processor, 2.2 GHz, and 2G of RAM.

Quantitative variables were analyzed using descriptive statistics. Unless otherwise indicated, data are presented as mean ± standard deviation. Qualitative variables were described as frequencies and percentages.

To evaluate the association between gender and outcomes of healing at 30 weeks, Fisher’s exact test was used. The wound contraction per week (WCw) was calculated as the baseline wound area (Ai) − final wound area (Af) ÷ the number of weeks.28

owm_0316_buzzi_equation

The percentage reduction in wound area per week (%RWAw) was calculated as follows:

owm_0316_buzzi_equation2

To compare the groups defined by healing within 30 weeks, the quantitative variables were analyzed using Student’s t-test, and the nonparametric variables were analyzed using the Mann-Whitney U test. Time to complete ulcer healing was measured as the number of days from the start of treatment to the date a patient achieved complete wound closure. Wound healing was observed on a weekly basis, and the time until complete healing was estimated by calculating a cumulative frequency chart. P <0.05 was considered to be statistically significant.

 

Results

The participants’ flow through the study is presented in Figure 1. Of the 109 subjects enrolled, 25 did not meet the inclusion criteria. Of the remaining 84 participants enrolled into the treatment phase, 43 withdrew before study completion. The main reasons for withdrawal were lost to follow-up (16), medical judgment (2), failure to attend >3 scheduled visits (17), violation of the protocol (5), and death (3). Three (3) patients died from complications due to diabetes (eg, DFU, acute myocardial infarction, kidney failure, and stroke). Forty-one (41) patients completed the trial and were included in the analysis.

Baseline characteristics. Patient demographics and ulcer characteristics at baseline and after the 30-week treatment are presented in Table 2. Of 41 patients treated with C. officinalis hydroglycolic extract, 17 (41%) were female and 24 (59%) were male. The average age of the patients was 62 (range 44–82) years. Laboratory clinical tests showed 35 (80.5%) were hyperglycemic with an average blood glucose level of 152.9 ± 76.01 mg/dL (range 82–395 mg/dL). Glycated hemoglobin was in the range of 6.4%. Serum albumin and hemoglobin levels at baseline of the patients who achieved total wound closure were 4.03 ± 0.49 g/dL and 12.9 ± 1.37 g/dL, respectively, which were not statistically different (P = 0.509, Student’s t-test) from those that did not achieve complete wound healing (albumin = 4.24 ± 0.32, hemoglobin = 12.7 ± 1.75 g/dL). medium_owm_0316_buzzi_table2

Most patients (34) exhibited Wagner type 1 ulcers (82.9%), which were generally located in the plantar, lateral face, and malleolus regions. All DFUs were characterized as chronic lesions because they were present an average of 71.1 ± 41.7 (range 17–156) weeks before the onset of this study. The baseline wound area was 8.68 ± 8.55 cm2 (range 1.2–43.1 cm2). The biogram/antibiogram tests from the swabs collected from the wound beds showed 31 (76%) of the ulcers were either colonized or infected. Twenty (20) DFUs (48.8%) showed high levels of bacteria ( >1 × 106 CFU) and were considered infected. The predominant clinical signs of the infected wounds were odor, exudate, erythema, and edema. Eleven (11) DFUs (26.8%) were colonized with low bacteria level and were not considered infected. Staphylococcus aureus was the predominant pathogen (11, 27%), followed by Pseudomonas sp. (4, 10%), Klebsiella sp. and Escherichia coli (3, 8% each). Of the 33 patients with colonized DFUs at the beginning of the study, 21 were treated with ciprofloxacin 500 mg twice daily for 30 days and 12 with gentamycin 80 mg intramuscularly once a day for 10 days. Two (2) patients did not satisfactorily respond to antibiotics, so they were excluded from the study.

Ulcer area reduction and healing rate. The time-course analysis indicated a linear increase in the proportion of patients achieving complete would closure after 11–30 weeks of treatment (see Figure 2). Thus, a minimum treatment period of 10 weeks was required for complete wound closure, and the average healing time was approximately 15.5 ± 6.7 weeks. After 11 weeks of treatment, 22 (54%) of the wounds were completely healed, with an average reduction in the wound area of 64% and noticeable improvements in the ulcer appearance from the baseline, including an increase in epithelialization and reduction of exudate, fibrin slough, necrosis, edema, erythema, and odor. After 20 weeks, 28 wounds (68%) were completely healed; after 30 weeks, 32 patients (78%) achieved complete healing and the remaining 9 (22%) achieved an overall reduction in the wound area of 75%. Figure 3 illustrates the process of diabetic ulcer healing in 3 patients treated with C. officinalis hydroglycolic extract. Photographs of patients’ DFUs showed complete epithelialization without apparent excess of anomalous tissue such as keloids and hypertrophic scars. owm_0316_buzzi_figure2

Patients were separated into complete DFU closure (coDFU group) and incomplete DFU closure (inDFU group) after 30 weeks of treatment. The inDFU group had mean baseline DFU areas (mean 10.28 ± 6.91 cm2; median: 8.43 cm2) similar to those of the coDFU group (mean 8.23 ± 9.02 cm2; median 5.72 cm2) (P = 0.105, Mann-Whitney U test) (see Table 3). In contrast, the wound contraction rate for the coDFU group was 3-fold higher (54.4 mm2/week; range, 5.2–168.7 mm2/week) than for the inDFU group (16.1 mm2/week; range 2.3–64.3 mm2/week) (Mann-Whitney U test; P = 0.001) (see Table 3). When the individual healing rates were corrected for baseline DFU area, the rates were nearly 6-fold higher in the coDFU group (7.75 ± 3.37%) than in the DFU group (1.38 ± 0.69%) (Mann-Whitney U test; P = 0.001). owm_0316_buzzi_figure3

Ulcer infection and inflammation. After 30 weeks of treatment with C. officinalis hydroglycolic extract, the total number of colonized DFUs significantly decreased from 29 (70.7%) to 5 (12.1%) (χ2 test; P = 0.012; see Table 2). C. officinalis extract was equally effective against all pathogens identified in the DFUs. Accordingly, the proportion of wounds presenting with unpleasant odor at baseline (19, 46.3%) was considerably lower after 2 weeks and ultimately decreased to1 (2.4%) after completion of the treatment. Ulcer bed planimetry data showed a significant reduction of exudate, fibrin slough, and necrotic tissue after the treatment with C. officinalis hydroglycolic extract (χ2 test; P = 0.001) (see Table 2). 

owm_0316_buzzi_table3

Pain assessment. Data from the NPSQ showed patients had a positive DFS for neuropathic pain of 0.543 ± 0.213 at baseline. All patients had a positive score for tingling pain, numbness, and increased pain due to touch. After 30 weeks of treatment, the average score for neuropathic pain was positive (0.552 ± 0.200) with no significant differences from the baseline score. The average patient pain score at baseline was 6.953 ± 2.160; at the end of 30 weeks of treatment, this was significantly reduced to 0.767 ± 0.477 (χ2 test; P = 0.001).

No adverse events with the C. officinalis hydroglycolic extract were observed during the treatment.

 

Discussion

C. officinalis hydroglycolic extract for the treatment of DFUs was assessed by having patients use it in their regular life at home with minimal influence from hospital/outpatient interventions. All patients received their first treatment in clinic then performed care at home. 

A systematic review of the literature4 that included 10 clinical trials on the performance of standard DFU treatments reported completely healed wounds in an average of 24% of patients at 12 weeks and in 30% of patients at 20 weeks; these data are used to benchmark DFU wound treatment therapies. The current study demonstrated 54% of DFUs were completely healed at 11 weeks and 68% at 20 weeks using C. officinalis extract. The average healing time to complete reepithelialization was approximately 16 weeks. At the study’s conclusion (30 weeks), 78% of all wounds had achieved complete closure. However, it should be noted that most studies utilized an intent-to-treat analysis method and the current study did not, which limits the ability to compare the results.

Several therapies are currently used to treat DFUs, including topical active solutions and ointments, silver, alginate- and cellulose-containing dressings, acellular protein dressings, autologous platelet-rich plasma, and bioengineered cell-based tissue grafts.29 Table 4 shows the percentage of healed DFUs in several previous prospective studies with different treatments.4,5,30-37 A direct comparison of the healing rates between the studies conducted with different therapies is not possible because of the differences in the demographics, comorbidities, wound complexities, study endpoints, and data analysis methods used. In general, prospective, randomized controlled and retrospective studies involving advanced therapies such as acellular protein dressings, autologous platelet-rich plasma, and bioengineered cell-based tissue grafts for treating DFUs achieve complete healing in 45% to 68% of wounds in 12 weeks29; however, with commonly used methods of care, this rate is roughly 30%.4 The average percentage of completely healed wounds in the present study is comparable to these other effective therapies.owm_0316_buzzi_table4

The anti-inflammatory and healing properties of C. officinalis are associated with a greater diversity of bioactives, specifically triterpene alcohols, triterpene oligoglycosides, monoesters, triterpenoids, and flavonoids.18–22,38–41 Chromatographic analysis from the dichloromethane fraction of C. officinalis flowers demonstrated 9 anti-inflammatory bioactive triterpenoids (ie, faradiol-3-O-palmitate, faradiol-3-O-myristate, faradiol-3-O-laurate, arnidiol-3-O-palmitate, arnidiol-3-O-myristate, arnidiol-3-O-laurate, calenduladiol-3-O-palmitate, calenduladiol-3-O-myristate, and calenduladiol-3-O-laurate) are commonly found in many varieties of Calendula flowers.19,20,41 The hydroglycolic extract of C. officinalis used in the present study was rich in these compounds.

Generating new blood vessels in the wound is an essential component of the wound healing process. Observational cohort studies42,43 of diabetic neuropathic foot ulcers indicated angiogenic activity may be insufficient for promoting expansion of new blood vessels into the wound site and restricting the entry of inflammatory cells and oxygen supply. The wound healing properties of C. officinalis may be associated with the activation of angiogenesis in the injured tissue. In addition to the known anti-inflammatory and antiseptic activity of the C. officinalis extract, the authors of the current study suggest the components present in C. officinalis extract, such as the monoesters triterpenoids, are able to stimulate vascular growth in DFUs through the release of angiogenic factors, particularly the vascular endothelial growth factor (VEGF), thus promoting granulation, tissue growth, and epithelialization. Although the presence and activity of VEGF in the wound bed through sampling and analysis of ulcer fluids and tissues was not possible in this study, evidence from animal studies44,45 supports angiogenesis is stimulated in the presence of C. officinalis extract: one study44 used aqueous extract from C. officinalis flowers to show an induction of vascularization in the chick chorioallantoic membrane and one study45 used C. officinalis extract to demonstrate accentuated angiogenic activity in both chicken chorioallantoic membranes and rat cutaneous wound models.

The importance of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) to the integrity of the extracellular matrix (ECM), which tightly control its metabolism in the normal healing process, has been shown in cell culture study.46 These proteolytic enzymes have been shown in animal studies42 to act on ECM, enabling migration of cells into the wound site and penetration of new blood vessels, which results in the deposition of new ECMs and the formation of new tissue, facilitating reepithelialization and wound contraction. In diabetic ulcers, an excessive activity of MMPs on ECM and deficiency of TIMPs result in the destruction of new ECMs, which consequently always delays wound healing. In a preclinical study, C. officinalis extract was used to prevent ultraviolet irradiation-induced oxidative stress in rat skin and was observed to increase the activity and secretion of MMPs 2 and 9, which may be associated with procollagen synthesis, regulation of inflammatory responses, and rearrangement of damaged skin.47 Studies39,48 using cultures of human and mouse fibroblasts demonstrated extracts of C. officinalis stimulated fibroblast proliferation and cellular metabolism through an increase in mitochondrial dehydrogenase activity, suggesting that several compounds of C. officinalis, particularly monoesters and triterpenoids, possess synergistic action in the reepithelialization process. In the present study, the observed removal of necrotic tissue in favor of granulation tissue, significant reduction in bacterial counts, and rate of epithelialization suggest bioactive components of C. officinalis extract can stimulate the entire tissue repair process.

The observed reduction in unpleasant odor, edema, erythema, exudate, bacterial colonization, and necrotic tissue may be due to the anti-inflammatory and bacteriostatic properties of the C. officinalis extract. Extracts of C. officinalis have been shown in in vitro microbiology study49 to have bactericidal and fungicidal effects against microorganisms isolated from hospital patients. The extract used in the present study showed anti-inflammatory, antimicrobial, and wound healing properties, which corroborate with previous clinical and experimental studies using C. officinalis phytopreparations.49–52 The clinical benefits of C. officinalis hydroglycolic extract in healing DFUs may be due to its chemical constituents such as monoesters, triterpenoids, triterpene alcohols, triterpene oligoglycosides, and flavonoids, which may act synergistically to promote wound healing.

It also may be advantageous that C. officinalis hydroglycolic extract is an aqueous preparation that leaves no adherent solid residue on the wound bed. This facilitates the process of cleaning and dressing changes and minimizes friction and potential small trauma to the emergent granulation tissue. 

 

Limitations

This study was limited by the small number of patients meeting the inclusion/exclusion criteria, which prevented the adequate fractionation of subgroups with a more regular baseline wound size. The intent-to-treat method of analysis was unsuitable for this study because a large majority of dropouts and noncompliant events (n = 33) occurred in the early phase of the research (first 3 weeks), which may have drastically diluted the treatment effect observed over 30 weeks. However, not using the intent-to-treat analysis method increases the risk of overestimating the effect of treatment. 

 

Conclusion

In this patient population, the use of extract of C. officinalis hydroglycolic extract was associated with a high percentage (78%) of DFUs healed. Although not a direct comparison, the proportion of healed DFUs was 2 to 3 times higher than the reported benchmark for assessing topical treatments for DFUs. No adverse events occurred, and the percentage of wounds with an unpleasant wound odor and wound pain ratings were significantly reduced at the end of the treatment period. No adverse events occurred. A randomized, controlled trial with a greater number of patients is needed to confirm the clinical efficacy of C. officinalis extract for the treatment of DFUs. Although pain at baseline and final assessment was reported, it is not known whether reduced average pain scores were the result of treatment or from the wound being healed. 

 

Acknowledgments

The authors acknowledge Alvaro Quintas, MD from PUC-PR for his help on the project; Marcia Olandoski, PhD for the statistical analysis; and Luiz Carlos Pereira, MD, Ney Alencar, MD, and Sergio Tarlet, MD from the Department of Dermatology at the Hospital, Santa Casa de Misericórdia of Curitiba, for their expert dermatology advice. The authors also thank Maurício Centa, MD from Hospital São Lucas and the nurse team, especially Flavia Pontes, NA for helping with the patients.

 

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Potential Conflicts of Interest: Franciele de Freitas, NP provided consultancy services to Phytoplenus Bioativos

 

Dr. Buzzi is Research Director, Proamplus Clinical Research Advisory LTD, Pinhais, Paraná, Brazil. Ms. de Freitas is a nurse practitioner, Phytoplenus Bioativos S.A., Pinhais, Paraná, Brazil. Dr. Winter is a physician affiliated with Hospital da Santa Casa de Misericórdia de Curitiba, Department of Dermatology, Curitiba, Paraná, Brazil. Please address correspondence to: Marcelo Buzzi, PhD, Proamplus Assessoria Clínica Ltd, Pinhais, Paraná; email: marcelo.buzzi@gmail.com.

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