Evaluation of the effectiveness of Berberis integerrima Bunge root extract combined with spearmint essential oil in the treatment of acne vulgaris: A randomized controlled clinical trial

Acne vulgaris is one of the most common dermatological disorders. Berberis integerrima Bunge belongs to the Berberidaceae family. Several studies on different Berberis species in addition to B. integerrima have shown antimicrobial, antioxidant, and anti‐inflammatory effects. Spearmint essential oil also has antioxidant, antibacterial, and anti‐inflammatory activities. This study aimed to evaluate the clinical effectiveness of the topical combination of B. integerrima root extract and spearmint essential oil in the treatment of acne vulgaris.


| INTRODUC TI ON
Acne vulgaris is one of the most common dermatological disorders which mainly occurs in adolescents and young adults.This disorder can occur at any age.Pilosebaceous glands of the skin become swollen and closed in acne vulgaris.Symptoms of acne vulgaris include comedones and inflammatory lesions, such as papules, pustules, and nodules. 1 Acne vulgaris is an inflammatory disease with multiple causes.The most important factors that play a role in the development of acne are hyperkeratinization of follicles, incomplete keratinization, the proliferation of Propionibacterium acnes, increased secretion of sebum, increased production of androgens, and a variety of inflammatory mechanisms. 2,3Acne also may be induced by some medications, for example, anabolic steroids, corticosteroids, isoniazid, and lithium. 4 is important to understand that acne may have long-lasting and disturbing physical and psychological effects, including hyperpigmentation, scarring, depression, and anxiety.A major problem of this disease is the formation of permanent scars, which usually result in tissue destruction after manipulation. 5,6In addition, there are significant financial costs associated with acne. 4 Consequently, it can negatively affect the quality of life of patients as much as debilitating diseases such as diabetes and asthma. 7 general, acne can be controlled; however, it is rarely completely cured, and most patients require maintenance therapy with one or more medications.Permanent scars and pigment changes can be prevented by the early effective treatment of acne. 8It is possible to treat this disorder with a variety of drugs, including topical (e.g., topical retinoids, benzoyl peroxide, azelaic acid, and topical antibiotics) and oral (e.g., isotretinoin and oral antibiotics) medications.It is important to note that the drugs used have several side effects. 9,10Most guidelines advise topical treatments as the first line for mild to moderate acne.When the medicines are used topically, diseased area exposure increases and systemic absorption is reduced. 4,8[11] Antibiotic resistance is one of the most serious problems associated with acne treatment.Guidelines are currently being developed to combat antibiotic resistance. 3According to a report, antibiotic resistance in Propionibacterium acnes has increased from 20% in 1978 to 62% in 1996. 12There is currently overprescribing of oral antibiotics.Acne makes a major antibiotic consumption in young people.In England, acne accounts for the main part of antibiotic exposure between 11 and 21 years of age.To prevent the emergence of antibiotic resistance, limiting oral antibiotics is recommended.Developing effective topical treatments, avoiding prolonged use of oral antibiotics, prescribing topical treatments in combination with oral antibiotics, and continuing topical medications as maintenance treatment are approaches to reduce systemic antibiotic prescribing in acne. 8ere are numerous well-known treatments for acne as mentioned but antibiotic resistance remains challenging.Furthermore, monotherapy options are not promising for comprehensive acne treatment, Combinational topical therapy has many advantages compared to monotherapy. 4Therefore, finding nonantibiotic acne treatments is essential.As a result, potential herbal treatments can be investigated.
Berberis integerrima Bunge (Barberry; Zereshk in Persian) is a plant belonging to the Berberidaceae family.It grows in the Middle East and the central part of Asia, and it grows in most parts of Iran, especially in the North and Northeast. 13,14ere has been evidence for a variety of effects of this plant, including antioxidants, 15 antibacterial, 16 lipid-lowering, 13 antiinflammatory and antinociceptive effects. 17,18Berberine, the main alkaloid of this plant, is thought to be responsible for many of these effects. 19Furthermore, plant anthocyanins have antibacterial and anti-inflammatory properties. 20ntha spicata L. also known as spearmint, is a medicinal plant belonging to the Lamiaceae family.Carvone and limonene are two of its major components.The pharmacological properties of M. spicata extracts and essential oils have been investigated for various health benefits, including antioxidant, antiparasitic, antimicrobial, and antiinflammatory properties. 21cording to the effects of these two plants, it is possible that B. integerrima root extract and Spearmint essential oil, especially in combination (through different mechanisms of action), be effective in treating acne vulgaris.On the other hand, the clinical effect of these substances has not yet been investigated topically in treating acne.Therefore, this study investigated the clinical effect of the combination of Barberry root extract and Spearmint essential oil in topical form in the treatment of acne vulgaris.The barberry roots were washed carefully and air-dried for 1 month.After drying it was checked again to be cleared of any unwanted animal or fungi residue or soil pollutants.The clean roots were powdered using a hard electrical mill (mesh number 100) capable of chopping wood.Then, 3 kg of plant powder was extracted by a cylindrical percolator.After swelling up the sample in ethanol 96% for 2 h, it was transferred into the percolator, and after 3 days of maceration time, the extraction was started with a flow rate of 4 mL/ min for 7 days while the fresh solvent (ethanol 96%) was added from the top of the percolator with the same flow rate.The extraction was continued until the extract was exhausted.For this purpose, a few milliliters of the last percolate was evaporated to dryness until it showed no residue, which means the roots are completely extracted.The percolated extract was filtered and concentrated by a rotary evaporator (Heidolph, Germany) at 40°C and in low pressure (40 mBar), and stored in a refrigerator until use (199 g dry weight).

| MATERIAL S AND ME THODS
For the purpose of standardizing the barberry root extract, the polyphenol content was determined according to Folin-Ciocalteu's method as described before. 22Briefly, 20 μL of the diluted solution were mixed with 100 μL of Folin-Ciocalteau reagent, 450 μL of water, and made up to 2 mL with saturated sodium carbonate solution.The solution was left for 2 h at room temperature and in a dark environment.Following that, the absorption was measured at 765 nm.Gallic acid standard diagram formula was calculated as y = 0.1091x + 0.0768 (R 2 = 0.995).Finally, the total phenol content (TPC) of the extract was calculated as 11.15 ± 0.382 mg gallic acid equivalent per gram of dry extract.Spearmint was standardized using gas chromatography coupled with a mass detector (Agilent 7890A, Agilent 5975C, USA) with a caplillay GC column (HP-5, 30 m × 0.25 mm, 0.25 μm).The oven temperature was started from 70°C, held for 1 min, ramped by 4°C/ min up to 230°C, held for 1 min. 23The major components of the oil include carvone (80.5%), dl-limonene (12.3%), menthol (1.5%), and menthone (0.9%).
The tested topical solution contained a formulation of 5% Berberis root dried extract and 1% spearmint essential oil (BRES).To prepare it, 45 mL of diluted Berberis extract (6.4% in ethanol), 9 mL of propylene glycol, 0.6 mL of tween 20, 0.6 mL of spearmint essential oil, and distilled water up to 60 mL were mixed together.
Clindamycin 1% topical solution as standard drug was prepared by 0.6 g of clindamycin powder (Merck, Germany) in a solution of 45 mL ethanol, 9 mL propylene glycol, 0.6 mL of tween 20, and water up to 60 mL.
Participants were selected from patients referred to the Isfahan Skin Diseases and Leishmaniasis Research Center Clinic.Prior to participation in the study, each patient was interviewed in order to become familiar with the study in detail, and, if they agreed, their written consent was obtained.
The inclusion criteria for study patients were age above 12 years and having mild to moderate acne vulgaris on the face (cheek, chin, forehead, or nose).
Mild acne was defined as the presence of noninflammatory lesions (including closed or open comedones), and the number of papules, and pustules <10 without any nodules or cysts.Moderate acne was defined as the presence of noninflammatory lesions (including closed or open comedones) and the number of papules and pustules to be <20 without any nodules or cysts. 24,25ere were several exclusion criteria as follows: ( 1 Patients who met the inclusion criteria consecutively participated in the study after completing the consent form and were randomly assigned to one of the two groups; the drug group (herbal solution) or the control group (clindamycin solution).The block randomization method was used for randomization.In this way, each arrangement of the two groups was drawn in pairs in quadruple blocks.The blocks were numbered from one to the end.In order to select the blocks, a random number table was used.Based on the arrangement of that block, patients were divided into one of two groups according to the order of admission.
Patients' demographic and clinical information, including their age, gender, height, weight, duration of acne, comorbidities and medications were recorded.The patients in the drug and control groups were asked to apply the topical solutions of barberry extract/ spearmint essential oil and 1% clindamycin, respectively, twice a day until the full clearance of the lesions and for a maximum of 4 weeks.
They were instructed to wash the face with water, pat dry it, and apply a thin layer of the solution to the affected areas using the cotton.Also, the patients were instructed to avoid sunlight exposure as much as possible and to use sunscreen with an SPF of at least 30 during the intervention.In addition to full explanations for the patients about the method of consumption, timing, etc, the patients in both groups were asked to report any possible side effects.
Finally, using statistical analysis, the mean score of mGAGS and the mean number of lesions at the two mentioned times were compared between the drug and control groups.
The primary outcome measures included the change in the mGAGS score and the change in the number of lesions totally and separately for each type of lesion at the end of the fourth week, and the secondary outcome variable was the number of cases of completely treated lesions at the end of the fourth week.
To ensure the safety of the topical product, all patients included in the study were tested for skin sensitivity.In this test, some medicinal solution containing barberry and spearmint extract was rubbed on the back of the volunteer's arm and covered with an anti-allergic adhesive.On the other arm, only a blank adhesive was applied.The adhesives were removed after 24 h and the results were checked half an hour later.In this test, redness and swelling of more than 0.5 mm were considered a positive reaction (allergy), and the patient was excluded from the study.
The following formula was used to calculate the sample size.The α error was 5% and the β error was considered 20%.According to a study comparing the number of pustular acne lesions at the end of an intervention between two groups, 26 the values of standard deviation (δ) and mean (μ) were placed in the following formula: Therefore, to ensure the validity of the results, a minimum sample size of 15 individuals was considered in each group.
The statistical analysis was performed using SPSS software version 25.After determining the distribution of data with the Kolmogorov-Smirnov test, the appropriate statistical test was selected based on the data distribution pattern (normal vs. nonnormal).The chi-square test was used to compare the qualitative variables (gender, underlying disease, medications, and type of lesions).To compare the values within each group, paired samples t-test or Wilcoxon Signed rank test was used, while independent samples t-test or Mann-Whitney U test was applied for comparison of the values between the two groups at each time.In each case, p < 0.05 was considered statistically significant.

| RE SULTS
A total of 102 patients were surveyed for the study, of whom 87 met the inclusion criteria and 75 consented to participate.These people were randomly divided into drug and control groups.Thirty-eight people in the herbal solution group and 37 people in the clindamycin group were included in the study.During the study, 8 participants from the drug group and 7 participants from the control group were excluded from the study due to no return for revisit, and finally, 60 patients (30 in each group) completed the study (Figure 1).In all cases, the results of the sensitivity test were negative and none of the patients showed any signs of allergy.
In Table 1, the basic demographic and clinical characteristics of the two groups of patients are compared.As seen, there was no significant difference between the two groups in terms of these variables (age, gender distribution, BMI, disease, medications, and duration of acne).
Table 2 shows the changes in the outcome parameters during the intervention.As seen, in terms of all parameters, including The CONSORT flowchart of the study.the mGAGS score, the total number of lesions, and the number of comedones and papule lesions, although there was a significant decrease within both groups at the end of the intervention (Figures 2-8), there was no statistically significant difference between the two groups.It should be noted that due to the small number of pustules and nodules in the patients, statistical comparison between the two groups was not possible.(Figures 2-8).
Consequently, the statistical analysis did not include changes in these two types of lesions.
During the study, no complete treatments were observed in both the drug and control groups.
No side effects were reported by any of the patients during the study.

| DISCUSS ION
In this study, at the end of the intervention (fourth week), mGAGS scores, the total number of lesions, comedones, and papules decreased significantly compared to the beginning of the study for the group receiving herbal solution (barberry extract/spearmint essential oil).8][29] Berberine also reduces oxidative stress by reducing ROS production. 30rious species of barberry and berberine have shown antimicrobial properties in several studies.Berberine has antibacterial activity and enhances macrophage bactericidal activity by increasing AMPK (AMP-activated protein kinase) signaling. 27Other suggested mechanisms for berberine's antibacterial properties include damage to the cell membrane, inhibition of protein and DNA synthesis, 31 inhibition of RNA transcription, 32 and inhibition of bacterial cell wall peptidoglycan synthesis. 33study that examined the antibacterial activity of different fractions of the hydroalcoholic extract of B. integerrima root identified four alkaloids that showed the greatest antibacterial activity against Brucella abortus including jatrorrhizine, berberine, palmatine, and columbamine.The antibacterial effect of jatrorrhzine (15 μg/mL) and columbamine (15 μg/mL) was comparable to streptomycin (10 μg/ mL) as a standard treatment. 166][37] Berberis aquifolium has been effective against P.
acnes and S. aureus and Coagulase-negative Staphylococcus aureus (CONS) isolated from skin lesions of individuals suffering from acne. 38,39 has been confirmed that berberine has antiandrogenic properties in both animal 40 and human 41 studies.The following mechanisms have been suggested for the antiandrogenic properties of berberine based on a review study 42 : increasing the level of sex hormone binding globulin (SHBG), leading to the reduction of free androgen levels; inhibition of androgen receptor signaling; and inhibition of androgen synthesis by interacting with aldo-keto reductase family 1 member C3 (AKR1C3), a key steroidogenic enzyme in humans.Furthermore, berberine reduces sebum production by suppressing sebaceous gland lipogenesis which may be contributing to its anti-acne effects. 43 the best of our knowledge, there are no animal and clinical studies on the effects of topical extracts of Berberis species on acne vulgaris.However, there are few animal studies which have shown the positive effects of topical barberry extracts on other disorders, including psoriasis, 44 uveitis, 45 and paw edema 46 showing the potential benefits of this plant in topical use consistent with our results.Furthermore, we found only two clinical studies evaluating the effectiveness of B. vulgaris, another plant in Berberidaceae family, in the treatment of acne vulgaris.In a double-blind, randomized, controlled clinical trial, the anti-acne effect of oral capsules containing aqueous extracts of B. vulgaris dried fruits was compared with that of placebo.According to the results, inflammatory and noninflammatory lesions as well as total lesions decreased by about 44% in the treatment group.In addition, no side effects were reported. 47In another clinical trial, fresh fruit juice from B. vulgaris was administered orally to 38 patients with mild to moderate acne for 30 days.In the treatment group, inflammatory lesions and total lesions were significantly reduced compared to the placebo group. 48Therefore, the results of these two studies are in agreement with our results.Nevertheless, our study examined B. integerrima's effect in comparison with standard treatment (clindamycin) and demonstrated that this plant had a similar therapeutic effect to such a known treatment.
Furthermore, it offers a topical formulation of the herbal extract which is likely to have fewer side than the oral form.
It is noteworthy that although we used a slight amount of M. spicata essential oil in our formulation, yet some observed effects might be attributable to this fraction, particularly its antimicrobial and hormonal effects.A number of in vitro studies have shown the antibacterial activity of M. spicata essential oil against a variety of bacterial strains, including S. aureus and S. epidermidis.A clinical trial showed that consumption of Menta spicata tea significantly decreased free testosterone and increased luteinizing hormone, follicle-stimulating hormone, and estradiol.Spearmint was proposed as an antiandrogenic treatment for mild hirsutism. 53other clinical trial with more volunteers and a longer duration confirmed these results. 54In addition to the mentioned benefits, one of the disadvantages of mint essential oil is the possibility of its evaporation during storage in warm weather conditions and the reduction of its expiration time.Another disadvantage of essential oils use is their special smell, which may not be pleasant for some patients.

| CON CLUS ION
In patients with acne vulgaris, topical application of the combination of B. integerrima root extract and spearmint essential oil reduces the severity and number of lesions.Therefore, this extract can be considered as a natural remedy for the management of acne vulgaris.In ) Receiving systemic anti-acne treatment during the last 3 months, (2) receiving topical anti-acne treatment during the last month, (3) receiving systemic antibiotics during the last month, (4) receiving antiandrogen drug during the last 3 months, (5) hepatic or renal disorder, (6) endocrine diseases (e.g., diabetes mellitus, hirsutism, polycystic ovary syndrome [PCOS], and thyroid disorders), (7) receiving oral contraceptives during the last month, (8) other skin disorders or infections, (9) pregnancy, (10) lactation, (11) consumption of any drugs that induce acne, and (12) abuse of alcohol and any other abused substances.

A
visioface device was used to photograph patients' faces prior to the beginning of the mentioned interventions and at the end of the fourth week.The number and type of lesions as well as the score of the modified Global Acne Grading Scale (mGAGS) were determined in each patient and documented.The mentioned scale is based on the location of acne and the type of lesion, and finally, the sum of the scores of different areas of the face is considered as the overall score.Acne severity, type, and the number of lesions were determined by a dermatologist.
solution and clindamycin, for all variables and studied factors.These results show a similar effect of the combination of barberry root extract and spearmint essential oil with clindamycin on reducing the number and severity of acne lesions.In terms of the side effects, no cases were reported by any of the patients during the study, showing the potential safety of the herbal product in topical use, including no allergic or irritating reactions.It is important to note that the number of research articles and scientific findings related to acne treatment regimens has continued to rise.Combination therapy, which addresses several pathogenic factors at the root of the condition, is the most recent development in the treatment of acne.Even though our knowledge of the causes of acne has grown significantly over the past 20 years, numerous attempts have been made to create novel treatments.Many studies have been conducted on acne by researchers, but only a small number of treatments have successfully passed clinical trials and are currently available to consumers. 4Although many studies have been conducted on the antiinflammatory, antimicrobial, and lipid and blood sugar lowering effects of B. integerrima, based on our research, this is the first clinical study conducted on the effect of topical extract of B. integerrima root on acne treatment.As mentioned in the introduction, the pathogenesis of acne is multifactorial.Inflammatory factors and mechanisms are among the most important causes of acne.Barberry and berberine alkaloids have been extensively studied for their anti-inflammatory effects.
extract of Berberis vulgaris leaves and roots were investigated in another study published recently in 2022.Both extracts inhibited the growth of Staphylococcus aureus, Staphylococcus enteritis, and Escherichia coli.Finally, B. vulgaris leaves and roots have been proposed as a natural source of powerful antioxidants and antibacterials.

Parameter Drug group (n = 30) Control group (n = 30) p-Value
Baseline demographic and clinical characteristics of study patients.The obtained results of the evaluated variables and their comparison between the groups.
TA B L E 1