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AProspective,DescriptiveStudytoAssesstheClinicalBenefitsofUsingCalendulaofficinalisHydroglycolicExtractfortheTopicalTreatmentofDiabeticFootUlcers

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A Prospective, Descriptive Study to Assess the Clinical Benefits of Using Calendula officinalis Hydroglycolic Extract for the Topical Treatment of Diabetic Foot Ulcers Marcelo Buzzi, PhD; Franciele de Freitas, NP; and Marcos Winter, MD

AbstractDiabetic 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 eval-uate 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 evalu-ated 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 heal-ing 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 reduc-tion 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.

Keywords: clinical study, diabetes complications, Calendula, diabetic foot, wound healing

Index: Ostomy Wound Management 2016;62(3):8–24

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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CALENDULA OFFICINALIS EXTRACT TO TREAT DIABETIC FOOT ULCERS

Diabetic foot syndrome is a common and severe compli-cation 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 heal-ing 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 foot-wear 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 amputa-tion is skin replacement therapies. Because skin replacement therapy is expensive and not widely available, more effective treatments for chronic DFUs are desperately needed for pa-tients 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, hy-perbaric oxygen, skin grafts, electrical and magnetic stimula-tions, 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, espe-cially with the new extraction methods designed to optimize their yield, purity, and bioactivity (eg, anti-inflammatory, wound healing, and antitumor properties).11–15 A prospec-tive, randomized controlled trial16 with 37 patients with dia-betes demonstrated topical kiwifruit extract reduced the size of DFUs by ~50% in 3 weeks compared to patients who re-ceived 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 sil-ver 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 poten-cies.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, ru-tin, kaempferol, and narcissine). According to animal stud-ies,18–22 the monoesters triterpenoids (ie, isopropyl myristate, palmitate, and laurate) are considered the most effective top-ical 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 PurposeA 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 com-plete wound closure and during a 30-week follow-up period to monitor the long-term effects of the treatment.

Methods and ProceduresParticipants. The study was conducted between May

2012 and December 2013 in the outpatient unit of the De-partment of Dermatology, Hospital da Santa Casa de Mi-sericórdia de Curitiba, Pontifícia Universidade Católica do

Key Points• The foot ulcers of 41 patients with diabetes mellitus

(DM) were treated with topical Calendula officinalis for a maximum study period of 30 weeks.

• C. officinalis flowers are used in numerous over-the-counter botanical preparations for external use as an anti-inflammatory wound healing agent.

• After 30 weeks of care, 78% of ulcers were healed (av-erage time to healing 15.5 weeks) and the percentage of ulcers assessed as infected was significantly lower.

• Despite important study limitations, the results suggest controlled clinical studies to evaluate the safety, effec-tiveness, and efficacy of this treatment for the manage-ment of foot ulcers in persons with DM are warranted.

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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 exclu-sion 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.

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 his-tory, 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, nutri-tional status, neuropathic pain assessment,23 and blood tests) also was recorded. Blood test results included glucose levels, glycated hemoglobin, serum albumin, blood count, erythro-cyte 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 hydro-glycolic 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 compound-ing pharmacies using commercial ingredients. The certifi-cated analysis of the C. officinalis hydroglycolic extract in-cluded the following compounds from dichloromethane fraction: β-amyrin (6.7%), lupeol (4.7%), ψ-taraxasterol

(8.1%), calenduladiol monoesters (5.5%), arnidiol monoes-ters (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. of-ficinalis 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 ap-plied. Patients were provided cushioned footwear, diabetic boots, crutches, and wheelchairs to offload the affected areas. None of the patients used additional wound healing medica-tion, phytopreparation, hydrogels, hydrocolloids, or support-ive therapy (ie, electrotherapy, vacuum therapy, laser therapy, phototherapy). The nursing team instructed patients or their caregivers to use sterile gauze dressings after each C. offici-nalis 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 cush-ioned 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 previ-ously.24 Photographs of each ulcer were taken with a Sony

Table 1. Inclusion and exclusion criteria

Inclusion criteria Exclusion criteria

• Age 18–90 years • Active Charcot foot

• Negative history of an allergy to Calendula officinalis or any plant in the Asteraceae family

• Non-neuropathic ulcers

• Able to provided written informed consent • Evidence of skin cancer within or adjacent to the target site of the ulcers

• Neuropathic ulcer as determined by clinical evaluation with a surface area between 0.5–45 cm2

• Osteomyelitis requiring treatment

• Ulcers without sinus tract, tendon, capsule, or bone exposure at baseline

• Cellulitis, osteomyelitis, gangrene, or deep tissue infection

• Adequate glycemic control • Pregnant women

• Able to visit the outpatient unit • Receiving or history of receiving systemic (oral/intravenous) corticosteroids, immunosuppressants, radiation, or chemotherapy within 4 weeks of study entry

• Adequate blood supply to the target vascular site • History of using C. officinalis extract on the ulcer site <12 weeks before the screening visit

• Able to tolerate sharp debridement when necessary

• Able to follow the strict protocol recommendations

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DSC-H1 digital camera (Sony USA, New York, NY, USA) at every nurse visit. Image capture was standardized using a tri-pod 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 ana-lyzed 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 dif-ferent tissue types such as granulation, epithelialization, fi-brin 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 ex-udate was confirmed by the appearance of moist gauze dur-ing dressing changes. The presence of odor was noted. The ulcer was classified according to the Wagner Grading Sys-tem,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, ede-ma, 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. of-ficinalis 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 guid-ance 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 imagin-able = 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 dis-criminant function score (DFS). DFS <0 predicted no neuro-pathic 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 evalua-tions and nursing. Data were analyzed by importing Access files to the database to the Statistical Package for Social Sci-ences (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 de-scribed as frequencies and percentages.

To evaluate the association between gender and out-comes 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

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

To compare the groups defined by healing within 30 weeks, the quantitative variables were analyzed using Stu-dent’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.

ResultsThe 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 comple-tion. The main reasons for withdrawal were lost to follow-up (16), medical judgment (2), failure to attend >3 sched-uled 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

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stroke). Forty-one (41) patients completed the trial and were included in the analysis.

Baseline characteristics. Patient demographics and ul-cer characteristics at baseline and after the 30-week treat-ment 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 com-plete wound healing (albumin = 4.24 ± 0.32, hemoglobin = 12.7 ± 1.75 g/dL).

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 col-lected 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 bacte-ria 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 ex-cluded from the study.

Ulcer area reduction and healing rate. The time-course analysis indicated a linear increase in the proportion of pa-tients 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 clo-sure, 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 epitheliali-zation without apparent excess of anomalous tissue such as keloids and hypertrophic scars.

Patients were separated into complete DFU closure (coD-FU 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).

Ulcer infection and inflammation. After 30 weeks of treat-ment 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 ex-tract 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

Figure 1. Study flow chart.

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lower after 2 weeks and ultimately decreased to1 (2.4%) af-ter 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 hydro-glycolic extract (χ2 test; P = 0.001) (see Table 2).

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. of-ficinalis hydroglycolic extract were observed during the treatment.

DiscussionC. officinalis hydroglycolic ex-

tract for the treatment of DFUs was assessed by having patients use it in their regular life at home with minimal influence from hospital/outpatient interven-tions. All patients received their first treatment in clinic then per-formed care at home.

A systematic review of the literature4 that included 10 clini-cal 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 approxi-mately 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 meth-

od and the current study did not, which limits the ability to compare the results.

Several therapies are currently used to treat DFUs, includ-ing topical active solutions and ointments, silver, alginate- and cellulose-containing dressings, acellular protein dressings, au-tologous 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 treat-ments.4,5,30-37 A direct comparison of the healing rates between the studies conducted with different therapies is not possible

Table 2. Patient demographics and ulcer characteristics at baseline and end of treatment

Baseline 30 weeks P valuea

Ulcer variables

Ulcer type, n (%) Wagner I 34 (82.9%) 9 (21.9%) 0.012

Wagner II 7 (17.1%) 0 (0.0%) 0.001

Ulcer location, n (%) Plantar region 7 (17.1%) 1 (2.4%) 0.001

Amputation stump 5 (12.2%) 2 (4.9%) 0.035

Calcaneus 6 (14.6%) 1 (2.4%) 0.015

Hallux 3 (7.3%) 0 (0.0%) 0.001

Phalanges 1 (2.4%) 0 (0.0%) 0.001

Lateral face region 7 (17.1%) 0 (0.0%) 0.001

Dorsal region 5 (12.2%) 1 (2.4%) 0.022

Malleolus region 6 (14.6%) 4 (9.8%) 0.045

Ulcer duration (weeks) Median (range) 65.0 (17.1–156.4)

Ulcer area (cm2) (Mean ± SD) 8.68 ± 8.55 0.57 ± 1.68 0.025

Tissue type of ulcer bed

Epithelium 5.1% 85.4% 0.001

Granulation 65.9% 29.3% 0.001

Minimal exudate 42.4% 2.4% 0.001

Moderate exudate 50.3% 1.5% 0.001

Fibrin slough 56.1% 4.8% 0.001

Necrosis 14.6% 0.0% 0.001

Unpleasant odor 46.3% 2.4% 0.001

Edema 36.6% 2.4% 0.001

Erythema 29.3% 4.8% 0.001

Ulcer microbiology Colonized DFUs 26.8% 14.6% 0.012

Infected DFUsa 48.8% 2.4% 0.001

Staphylococcus aureus

26.8% 5.6% 0.010

Pseudomonas sp. 9.8% 2.4% 0.011

Klebsiella sp. 7.3% 0.0% 0.001

Escherichia coli 7.0% 1.0% 0.024

Others 17.1% 3.1% 0.015a high bacterial levels (>1 × 106 CFU ). P values are from Fisher’s t-test, P <0.05. DFU=diabetic foot ulcerDO N

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because of the differences in the demo-graphics, comorbidities, wound complex-ities, 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 pres-ent study is comparable to these other ef-fective therapies.

The anti-inflammatory and healing properties of C. officinalis are associated Figure 2. Cumulative proportion ulcers healed over time (weeks).

Figure 3. The clinical course of 3 patients with diabetic foot ulcers treated with the Calendula officinalis hydroglycolic extract.

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with a greater diversity of bioactives, specifically triterpene alcohols, triterpene oligoglycosides, monoesters, triterpe-noids, 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, faradi-ol-3-O-laurate, arnidiol-3-O-palmitate, arnidiol-3-O-my-ristate, arnidiol-3-O-laurate, calenduladiol-3-O-palmitate, calenduladiol-3-O-myristate, and calenduladiol-3-O-lau-rate) 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 co-hort studies42,43 of diabetic neuropathic foot ulcers indicated angiogenic activity may be insufficient for promoting expan-sion of new blood vessels into the wound site and restrict-ing 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 stim-ulate vascular growth in DFUs through the release of angiogenic factors, particu-larly the vascular endothelial growth factor (VEGF), thus promoting granulation, tis-sue 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 stud-ies44,45 supports angiogenesis is stimulated in the presence of C. officinalis extract: one study44 used aqueous extract from C. of-ficinalis flowers to show an induction of vascularization in the chick chorioallantoic membrane and one study45 used C. offici-nalis extract to demonstrate accentuated

angiogenic activity in both chicken chorioallantoic mem-branes 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 de-struction 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,

Table 3. Wound contraction (WCw) and percentage reduction in wound area per week (%RWAw)

Variable Healing in 30 weeks

N Mean Median Lowest value

Highest value

SD P valuea

Wound contraction – WCw (cm2/week) coDFU 32 0.543 0.366 0.053 1.687 0.443 0.036

inDFU 9 0.161 0.126 0.023 0.643 0.191

Percent reduction wound area/week (%RWAw)

coDFU 32 7.75 6.90 3.37 16.28 3.58 0.001

inDFU 9 1.38 1.41 0.33 2.45 0.69a Nonparametric Mann-Whitney U-test, P < 0.05; DFU = diabetic foot ulcer; coDFU=patients with complete DFU closure; inDFU=patients with incomplete DFU closure; SD=standard deviation

Table 4. Reported proportion of healed diabetic foot ulcers

Study Treatment Follow-up period (weeks)

Number of participants

Proportion of healed ulcers (%)

Current study COHE 20 43 68

Current study COHE 11 43 54

Wieman et al32 rhPDGF 20 382 50

Veves et al37 BHSE 12 208 56

Caravaggi et al35 ADG 11 79 67

Marston et al31 HDSS 12 314 30

Driver et al33 PRPD 12 72 68

Reyzelman et al30 AM 12 86 69

Edmonds et al5 BHSE 12 72 51

Uccioli et al36 ADG 12 180 24aMargolis et al4 ST 12 26,600 24aMargolis et al34 PRPD 32 26,600 51aRetrospective meta-analysis study. COHE=Calendula officinalis hydroglycolic extract; ST=standard treatment; AM=acellular matrix; ADG=autologous dermal grafts; HDSS=human dermal skin substitute; BHSE=bilayered human skin equivalent; rhPDGF=recombinant human platelet-derived growth factor; PRPD=platelet-rich plasma derivatives

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and rearrangement of damaged skin.47 Studies39,48 using cul-tures of human and mouse fibroblasts demonstrated extracts of C. officinalis stimulated fibroblast proliferation and cellu-lar metabolism through an increase in mitochondrial dehy-drogenase 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 fa-vor of granulation tissue, significant reduction in bacterial counts, and rate of epithelialization suggest bioactive com-ponents of C. officinalis extract can stimulate the entire tissue repair process.

The observed reduction in unpleasant odor, edema, ery-thema, 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 bac-tericidal and fungicidal effects against microorganisms iso-lated 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 phytoprepara-tions.49–52 The clinical benefits of C. officinalis hydroglycolic extract in healing DFUs may be due to its chemical constitu-ents 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 hydroglycol-ic 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.

LimitationsThis 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 drop-outs and noncompliant events (n = 33) occurred in the early phase of the research (first 3 weeks), which may have dras-tically diluted the treatment effect observed over 30 weeks. However, not using the intent-to-treat analysis method in-creases the risk of overestimating the effect of treatment.

ConclusionIn this patient population, the use of extract of C. officina-

lis hydroglycolic extract was associated with a high percent-age (78%) of DFUs healed. Although not a direct compari-son, 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 treat-

ment 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 as-sessment was reported, it is not known whether reduced av-erage pain scores were the result of treatment or from the wound being healed. n

AcknowledgmentsThe 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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