The Antimicrobial and Healing Effect of Scrophularia striata Boiss Hydroalcoholic Extract on First- and Second-Grade Pressure Wounds in Patients with Brain and Spinal Cord Injury: A Randomized Clinical Trial

Materials and Methods This double-blind clinical trial study was performed on grade 1 and 2 pressure wounds in 120 patients with cerebral-spinal cord lesions. The patients were randomly divided into four groups (n = 30). Topical treatments in all groups were performed twice a day. These groups included experiment 1 (SHE + phenytoin), experiment 2 (SHE + SHE), control (phenytoin + phenytoin), and placebo (eucerin + phenytoin). After evaluating the effect of SHE on wound healing, its antibacterial activity was determined by the standard agar well diffusion method. Results Patients in each group in this study did not significantly differ in demographic and clinical variables. Complete wound healing by the 10th day of the intervention occurred in 63%, 100%, and 27% of patients in experimental 1, experimental 2, and control groups, respectively. In contrast, the placebo group had no complete wound healing until the 10th day. Topical application of SHE, twice a day in the experimental 2 groups, had a higher potency to heal wounds and reduce the duration of complete wound healing in patients compared with other groups. Conclusion SHE, as a novel treatment option, has good potential to accelerate the healing of first- and second-degree pressure wounds in patients with brain-spinal cord injuries.


Introduction
Pressure ulcers are a common skin injury among patients with brain and spinal cord lesions and permanent movement disorders. In addition to causing pain and decreased function, this complication increases the risk of bone and blood infections, lengthens the hospital stay, and necessitates additional surgical procedures [1][2][3]. As a result of constant pressure placed on the skin for an extended period, pressure ulcers can destroy the skin and the underlying tissues, ultimately leading to tissue necrosis and loss of nutrition and oxygen [4]. Patients in long-term care facilities, rehabilitation centers, and intensive care units (ICUs) are susceptible to pressure ulcers at up to 23% [5]. According to the Agency for Healthcare Research and Quality (AHRQ), over 2.5 million Americans sufer from pressure ulcers, and 60,000 people die of pressure ulcers each year [5]. Medical management of pressure ulcers costs the US healthcare system between $9.1 billion and $11.6 billion annually because of increased healthcare utilization [5]. Te prevalence of pressure ulcers in European hospital inpatients is above 18%, with only 10% of patients receiving appropriate preventive care [6]. In Iran, the prevalence of pressure ulcers in hospital patients is 44% [7,8]. Consequently, preventing and treating pressure ulcers can reduce sufering, long-term hospitalisation, patient mortality, and healthcare costs [7].
Tere are many sites where pressure ulcers can form, including the tibia, scapula, scapular, lumbar spine, iliac crest, sacrum, elbows, heels, and ankles [9]. Te techniques include changing the patients' conditions, not scrubbing the wound, applying dressings, lubricants, alginates, or supportive dressings to protect the wound from friction, nutritional support, and physical therapy measures, and rehabilitation to assist patients in walking again [10,11]. Furthermore, traditional medicine has been considered in studies today as a means of preventing and treating pressure ulcers due to the side efects of chemical drugs. Terefore, using wax, honey, some plant extracts, and oils such as those from sunfower, olive, and borage roots is recommended for treating pressure ulcers [8,12]. Te use of natural antibiotics, such as drugs and antibiotics derived from plants, is regarded as efcacious [13]. In the western part of Iran, the Zagros Mountains are home to one of the most important medicinal plants, Scrophularia striata, also known as fgwort. In Ilam province (West Iran), this plant species has been used for treating many diferent illnesses for many years, despite not knowing its chemical composition [14,15]. In this sense, Scrophularia striata is a plant species that heal wounds and prevents bacterial infections [16,17].
Te genus Scrophularia includes 300 species of Scrophularia striata [18]. Plant extracts of this plant contain favonoids, monoterpenes, and coumarins, which are antioxidants and antibacterials. Scrophularia striata reduce edema, cell infltration, and proliferation of T lymphocytes by inhibiting the production of prostaglandin E2, which is responsible for producing interferon-gamma and tumour necrosis factors [18,19]. Additionally, phenolic acids with antibacterial properties are found in this plant, contributing to its efectiveness as a wound healer [20]. It has been demonstrated that Scrophularia striata have properties that stimulate fbroblast regeneration, antioxidant activity, and anti-infammatory activity and promote faster wound healing, angiogenesis, and vasodilation [21]. In samples treated with Scrophularia striata extract, an increase in fbroblast cells and collagen fbres with the formation of epidermis and dermis has been observed at the wound site [22].
Since pressure ulcers are considered a life-threatening condition for patients with movement disorders caused by brain and spinal cord lesions, using various treatment and care methods, especially herbal medicines, is vital to quickly and completely prevent and treat pressure ulcers. In lab experiments, Scrophularia striata have shown properties to be anti-infammatory, antibacterial, and antioxidant. In this study, we examined the efect of hydroalcoholic Scrophularia striata extract on healing frst-and second-grade pressure ulcers in patients with brain and spinal cord lesions.

Trial Design.
Tis randomized controlled and blinded clinical trial was conducted to evaluate the antimicrobial and healing efect of Scrophularia striata Boiss hydroalcoholic extract on frst-and second-grade pressure wounds in patients with brain and spinal cord injury. From December 2019 to October 2020, 140 survivors with brain and spinal cord lesions were admitted to the intensive unit care of Besat, Imam Hossein, Shahid Beheshti, and Sina educationalmedical centers afliated to Hamadan University of Medical Sciences and included by the available sampling method. Tis study complied with the Consolidated Standards of Reporting Trials (CONSORT) statement [23]. Te ethics committee approved this study of Hamadan University of Medical Sciences under the code IR.UMSHA.REC.1398.747 and was registered in the Iranian clinical trial database with the code IRCT20120215009014N328 and address http:// www.irct.ir. A Helsinki Declaration guideline was also followed in the conduct of the study. Te patient's consent was obtained directly after they were discharged from the ICU. Informed consent covered both study participation and consented to publish the fndings. In cases where the patient lacked decision-making capacity, the patient's legal guardian or healthcare proxy could consent on the patient's behalf. Te group of patients with their family members or friends was called the enrolled family. Additionally, the study was conducted by the Helsinki guideline.

Inclusion and Exclusion
Criteria. Inclusion criteria include no history of allergy to Scrophularia striata extract and its products, having frst-and second-grade pressure ulcers in at least 1 cm from the skin, age group of 15-75 years, no history of asthma, chronic obstructive pulmonary disease (COPD), hay fever and swelling of the nasal mucosa, skin sensitivity, lack of skin lesions preventing examining the pressure ulcers, nonuse of anti-infammatory drugs (glucocorticoids, NSAIDs) for similar symptoms or other symptoms (such as rheumatoid arthritis), and lack of concomitant participation in another clinical trial efective on treatment of pressure ulcers. Also, the patients were excluded with withdrawal from the study earlier than one week, discharge or the personal satisfaction of the patient or transfer of the patient to a center outside Hamedan, intolerance and allergy to Scrophularia striata extract during the study, local skin complications not allowing the use of test cream, concomitant and indirect use of antiinfammatory drugs (glucocorticoids, NSAIDs), forgetting or not using the extract 2 or 3 times a day (once negligible), patients with diabetes mellitus with diabetic ulcers and diabetic neuropathy, and third-grade pressure ulcers.

Sample
Size, Randomization, and Blinding. Te sample size was determined based on the study carried by Azhdarmehri et al. [28], including 120 patients. Te patients were divided into four groups of 30 people considering the frst type error of 0.1, the study power of 0.90, the diference in the score of pressure ulcer size up to 10 mm, and the standard deviation of 10. After selecting the patients based on the inclusion criteria and obtaining conscious written consent, the study samples were randomly divided into four groups (three experiment groups and a control group) of 30 people by quadruple random blocks ( Figure 1).

2.4.
Intervention. Experiment 1 group consisted of patients receiving 1% phenytoin topical cream every morning and 10% topical formulation of Scrophularia striata extract on pressure ulcers, 12 h later. In experiment group 2, the patients received 10% topical formulation of Scrophularia striata extract in the morning and 12 h later in the mentioned areas. In the control group, patients received 1% topical phenytoin cream every morning and 12 h later, and in the placebo group, they received 1% of phenytoin topical cream every morning and 12 h later and placebo or eucerin on the mentioned areas to the extent covering the radius of the pressure ulcers. To blind the study, the hydroalcoholic extract of Scrophularia striata and placebo were prepared by the research pharmacist and prepared in a tube and given to the researcher and the sample nurse (ulcers expert). Te tubes containing the herbal extract were marked by X, and the tubes containing the placebo were marked by Y, and only the researcher was aware of the contents of the tubes. Te examiner and nurse, the patient, and the statistical analyzer were unaware of any of the contents of the labelled tubes. To prepare the hydroalcoholic extract of Scrophularia striata, 1.5 kg of rhizomes and stems of this plant, after approval by the botanist in the herbarium of the Faculty of Pharmacy, were completely dried at room temperature 25°C in the shade and then formed into a dry powder by a mechanical mill. 300 g of dried plant powder was soaked in 70°ethanol solution on a shaker for 72 h until most of the active ingredients were extracted. Te resulting mixture was fltered through flter paper and placed in a rotary apparatus at 50°C, 60 rpm; then, the solvent was removed and placed under the hood in a Petri dish for another week to dry and prepare the fnal extract. Te fnal extract was approved by a pharmacist.

Data Collection.
During ten days of the study, in all 4 groups, the area of the pressure ulcers was measured every 24 h until complete wound healing. All patients in the study received routine care for pressure ulcers, such as changing positions every 2 h and using a wavy mattress. Tis study was performed on selected patients from the hospital's onset of grade 1 and grade 2 pressure ulcer symptoms. Before intervention, skin tests were performed on patients with a topical formulation of pressure ulcers. Tus, some of this herbal formulation was tested on 5 mm of the patient's forearm skin and examined 24 h later. Te intervention was stopped in case of any skin complication or allergy in the patient. Patient's demographic information, including age, gender, body mass index, occupation, education, and location, were recorded. A questionnaire was used to record the observations and diagnosis of the doctor and nurse to evaluate the extent of pressure ulcer healing during 10 days of the study. In this questionnaire, skin sensitivity to Scrophularia striata extract and ulcer size were recorded, measured daily and accurately at 8 am. Te ulcer size was measured using transparent paper and a permanent pen, and fnally, by scanning the sheets; the ulcer circumference size was interpreted by image j software in square millimetres. Te examinations were scored daily from 1 to 4 based on pressure ulcer grades.

Agar Difusion Susceptibility Tests.
Te susceptibility test was carried out by the standard agar well difusion method for various concentrations of the extract against common organisms found in infectious wounds. Soybean casein digest agar plates were inoculated by spreading 100 µl of Staphylococcus aureus and Pseudomonas aeruginosa suspensions at half McFarland (1.5 × 10 8 cfu/ml) bacterial concentration [1]. 10 mm diameter wells were punched in the plates by using a sterile stainless-steel borer and flled with extract dissolved in DMSO at 10, 5, and 1 mg/ml concentrations. 10 µg/ml clindamycin and ceftazidime solutions in water were used as a positive control against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. After incubating the plates at 37°C for 24 h, they were evaluated by measuring the inhibition zone diameters at 0.1 mm accuracy. Tis test was conducted in triplicate.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
MIC of the extract against Staphylococcus aureus and Pseudomonas aeruginosa was determined by using the macro dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [2]. Staphylococcus aureus and Pseudomonas aeruginosa suspensions inoculated soybean casein digest broth medium at half McFarland bacterial concentration to a fnal concentration of 1.5 × 10 6 cfu/ml. 20 mg/ml extract stock solution in DMSO was prepared. 5 ml of the extract stock solution was added to a test tube containing 5 ml inoculated broth medium to obtain a 10 mg/ml concentration. Serial dilution was continued in more than 11 test tubes to get extract solutions in the 0.003-10 mg/ml range. One test tube without adding Evidence-Based Complementary and Alternative Medicine the extract was used to show maximum bacterial growth, and another containing an uninoculated medium was used as proof for the aseptic technique. After incubation of the tubes at 37°C for 24 h, the concentration at which no growth was observed was determined as MIC [3]. Determination of MBC was performed by adding 100 µl from the constituents of the wells not showing microbial growth in the MIC test on agar plates. After incubation at 37°C for 24 h, the lowest concentration of the extract that did not form any colonies was determined as MBC. Tese tests were conducted in three separate experiments, and quantities selected as MIC and MBC in at least two experiments were reported.

Statistical
Analysis. Data analysis was performed by using Stata 23 software. Te chi-square test was used to compare the nominal variables and the independent t-test. Repeated measures analysis of variance, covariance, and ANOVA were used to compare the quantitative variables. A signifcance level was considered 5%.

Results
In this study, a total of 140 patients were selected. Twenty patients were excluded due to early withdrawal. Finally, 120 patients' information was divided into four groups and reviewed ( Figure 1). Te patients in the groups were not signifcantly diferent in terms of gender, age, occupation, education, marriage, and residence (P > 0.05). Table 1 shows the demographic characteristics of the patients. Body mass index in the patients of experiment group 1 was 24.60 ± 2.02, 24.13 ± 1 1.20 in experiment group 2, 23.96 ± 1.74 in the control group, and 23.97 ± 1.58 in the placebo group; this diference was not signifcant (P � 0.423).
Comparing the groups in terms of the reason for hospitalisation, most patients were hospitalised in experiment group 1 (53.3%) and the placebo group (63.3%) owing to spinal cord injury (SCI). Te patients in experiment group 2 (56.7%) and control (53.3%) were hospitalised mainly due to cerebrovascular accident (CVA). Te hospitalisation period of most patients in all four study groups was 7-12 days after the start of the study. Most of the patients had grade 2 ulcers in all four groups. Patients in the four groups were not signifcantly diferent regarding cause, location, period of hospitalisation, and type of ulcers (P < 0.05).

Patients' Profle of Clinical Characteristics in Four Study
Groups. Studying the mean ulcer size before and ten days after intervention in four groups, the results showed that there was a statistically signifcant diference between the mean ulcer size of four groups before intervention (P < 0.001) (Figure 2 and 3). Table 2 shows patients' clinical characteristics such as the case of hospitalisation, inpatient department, hospitalisation, and wound type in four groups of study. Terefore, the four groups were not identical regarding ulcer size before intervention (Table 3). Hence, analysis of covariance showed that there was a statistically signifcant diference between the mean ulcer size of four groups every ten days of intervention (P < 0.001) (Figures 2 and 3). Furthermore, repeated measures analysis showed that in all four groups, there was a statistically signifcant diference between the mean ulcer size on days 1-10 (P < 0.001) (Figures 2 and 3). In these groups, a decreasing process in ulcer size was observed during the study days. Te results of the ANOVA test and analysis of variance with repeated measures in comparison with the mean ulcer size before and after the intervention in four groups are presented in Tables 3 and 4. As the diagram shows, the decreasing process slope of the mean ulcer size in patients of experiment group 2 was steeper compared with experiment group 1, control, and placebo groups (Figure 3). According to this table, in the frst to ffth days of intervention, no complete ulcer healing was observed in any of the patients. By the tenth day of intervention, 63.3% of patients in experiment group 1, 100% in experimental group 2, and 26.7% in the control group had healing ulcers. During ten days of intervention, no complete recovery was observed in any placebo patients. Tere was a statistically signifcant diference between the distribution of research subjects in terms of complete ulcers healing during ten days of intervention in three experiment group 1, experiment group 2, and the control group (P < 0.001), and the process of complete ulcer healing in these three groups was observed during the observation. Studying the distribution of entire ulcer healing time in four groups indicated that the time of complete ulcer healing patients in experiment group 2 was less than in group 1 and the control group (Table 5). Also, the recovery occurred on the tenth and eighth days of the intervention in 50% of patients in experiment group 1 and group 2, respectively. Based on the semiparametric log-rank test, there was a statistically signifcant diference between patients' complete ulcer healing time in four groups (P < 0.001), and the full ulcer healing time in experiment group 2 was signifcantly lower than the other three groups.

Antibacterial Activity of the Extracts against Staphylococcus aureus and Pseudomonas aeruginosa.
Antibacterial activity of the extract of Staphylococcus aureus and Pseudomonas aeruginosa was investigated by the agar well diffusion susceptibility test (Figure 4). Table 6 shows the diameters of inhibition zones obtained for diferent extract concentrations along with 10 µg/ml clindamycin and ceftazidime solutions used as a positive control. Results of this test indicate concentration-dependent antibacterial activity of the extract against investigated microorganisms, along with higher antibacterial activity against Staphylococcus aureus. Evidence-Based Complementary and Alternative Medicine Table 7 shows estimated amounts of MIC and MBC. As can be seen, MIC and MBC are the same for both microorganisms, indicating the extract's high bactericidal potency. Tis test's results agree with the ones obtained in the agar difusion test and show the higher antibacterial activity of the extract against the Gram-positiveStaphylococcus aureus.

Discussion
Tis study aimed to determine the efect of hydroalcoholic extract of Scrophularia striata on the healing of grade 1 and 2 pressure ulcers in patients with brain and spinal cord lesions. Te results of daily examination of patients in terms of ulcer size during the intervention period showed signifcant test results in experiment group 1, experiment group 2, control group, and placebo group. Respectively, in experiment group 1, 63% of the subjects had complete ulcer healing by the tenth day of intervention. In experiment group 2, complete ulcer healing was observed for 100% of the subjects by the tenth day of the intervention. In the control group, for 27% of the subjects, complete ulcer healing occurred by the tenth day of the intervention. In none of the subjects, complete ulcer healing was observed in the placebo group until the tenth day of the intervention. Furthermore, the duration of complete ulcer healing of the patients in experiment group 2 was less than that of experiment group 1 and the control group. Also, the duration of complete ulcers healing in experiment group 2 was signifcantly shorter than in other three groups. Terefore, the percentage and process of ulcer healing were higher and faster in experiment group 2 than in other three groups.   Tey studied the healing efects of Scrophularia striata. According to Sharif et al., the Scrophularia striata plant improves episiotomy wound healing and facilitates faster healing so that it can be used to hasten episiotomy wound healing [24]. Scrophularia striata was also found to have antibacterial and wound-healing efects by Tanideh et al. in an animal study, indicating that the herb should be used when burn wounds are infected with P. aeruginosa [25]. Several antioxidant and larvicidal properties were observed for Scrophularia striata collected in the west of Iran [26]. Scrophularia striata have the potential to act as an antioxidant, but in reality, it can be concluded that the complex interaction of ecological and chemical processes which take place in the plant under such circumstances has resulted in the synthesis of diferent antioxidants compounds in the plant in diferent areas [15]. Te diferent glycopropenoids in Scrophularia species reduce edema, stop cell infltration, and have anti-infammatory properties [20]. Phenylpropanoid glycosides inhibit the activity of macrophages, thus inhibiting the production of infammatory chemical mediators and ultimately reducing infammation [21].
Furthermore, the presence of phenolic acids with antibacterial properties is another reason for the efectiveness of this plant in healing skin ulcers [15]. Stimulation of fbroblast growth and antioxidant and anti-infammatory properties of Scrophularia striata, along with stimulation of collagen production and faster ulcer contraction, angiogenesis, and vasodilation, confrm the efect of this plant in burn ulcer healing [16]. In specimens treated with Scrophularia striata extract, an increase in fbroblast cells and collagen fbres with the formation of epidermis and dermis was observed at the site of the ulcer [17]. Despite diferences such as method, target group, and dependent variable, a double-blind, randomized controlled trial indicated that compared to chlorhexidine disinfectant mouthwash, Scrophularia striata decreases plaque index, pocket depth, and probing bleeding in the short term but increases the number of Streptococcus mutans over a long time. Te fndings of this study demonstrate that Scrophularia striata have antimicrobial properties [27].

Conclusion
Tis study showed that using Scrophularia striata cream accelerates the healing of pressure ulcers in frst-and second-grade patients. Terefore, the use of Scrophularia striata is recommended to reduce the period of hospitalisation of patients following pressure ulcers and improve the quality of medical services and care.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.