Spray film-forming system formulation of Mitragyna speciosa as a diabetic wound healer

Abstract

Diabetic wounds are chronic complications in patients with diabetes mellitus characterized by an elongated proliferation period, leading to a longer wound closure time. The lack of topical remedies for diabetic wounds necessitates the development of formulations that effectively facilitate closure. Kratom (Mitragyna speciosa) leaves, containing polyphenolic compounds, have the potential to be developed into a film spray suitable for diabetic wound closure. This study aims to develop a film-spray formula of kratom leaves extracted using natural deep eutectic solvent (NADES) and to investigate its wound healing activity on type I diabetic rats. Polyphenol-rich NADES extracts were formulated with a film-forming base in concentrations of 2%, 3%, and 4% v/v. The sprays were assessed for pH, viscosity, drying time, and organoleptic properties and evaluated for their effectiveness in reducing wound diameter on streptozotocin-induced diabetic rats. All formulated sprays exhibited watery form, dark orange, and odor characteristics of kratom extract, and the stability test showed that no separation was observed. The wound healing activity evaluation revealed that the film spray containing 4% of kratom NADES extract on day 21 demonstrated significant healing effects on type I diabetic rats, including a reduction in wound diameter (83.99 ± 12.02%), a decrease in neutrophil cell count, and an increase in epithelial tissue thickness. Kratom NADES extract film-forming spray (4%) has the potential to shorten the closure time of diabetic wounds.

INTRODUCTION

The increasing prevalence of diabetes mellitus (DM) and its associated complications, including diabetic wounds, highlight the urgent need for effective treatments in wound care.[1] The growing number of diabetic patients has brought attention to the limitations of current wound management strategies, which mainly focus on blood glucose control and the use of antiseptics.[2,3,4,5] While these approaches can help prevent infection, they do not directly promote wound healing or closure.[6] This gap in treatment options has created a strong demand for new, more effective therapies, particularly those derived from natural sources.

Kratom (Mitragyna speciosa), a tropical plant native to Indonesia, has emerged as a promising candidate for addressing this issue.[7,8] This plant is traditionally used for various medicinal purposes, including pain relief, diarrhea, and postpartum care, and the leaf compress is used to treat wound.[9,10] The leaves of kratom contain a variety of secondary metabolites known for their bioactive properties, including antidiabetic,[11] antioxidant,[12] antibacterial, antimicrobial, and anti-inflammatory effects.[13] Studies have shown that phenolic compounds and flavonoids in kratom may contribute to faster wound healing by promoting blood clotting, stimulating angiogenesis, fighting infection, and accelerating tissue regeneration.[14]

The application of green extraction to obtain polyphenol-rich extracts based on natural deep eutectic solvent (NADES) combined with microwave-assisted extraction (MAE) technology has several advantages compared to organic solvents because it is selective for target metabolites, nontoxic, edible, economical, short extraction time, and nonflammable and has no adverse impact on the environment and humans.[15,16,17,18] The application of NADES and MAE for kratom leaf extraction can produce polyphenol-rich extracts that are safe to use as active ingredients for spray film preparations.

Spray film for diabetic wounds is an innovative dosage form specially formulated to form a thin film that covers diabetic wounds. Diabetic wounds have been treated with antiseptics to prevent secondary infections, whereas there has been little focus given to wound healing and closure.[19] This study presents a novel approach with the development of a wound-dressing film spray that effectively covers diabetic wounds by creating a protective film layer. Additionally, there have been no reports yet on the use of polyphenol-rich kratom leaf extract derived from NADES-MAE in a film-spray formulation to treat diabetic wounds. This research objective was to identify the potential use of kratom leaf extracts, particularly when prepared with NADES, as a novel treatment for improving wound healing, especially in individuals with diabetic conditions.

MATERIALS AND METHODS

Materials

Plant material

Kratom (M. speciosa) leaves were collected from Kota Bangun Village, Kutai Kartanegara Regency, East Kalimantan, Indonesia. The plant (specimen number: 002/BRN/07/2024) was identified and authenticated in the Laboratory of Dendrology, Faculty of Forestry, Universitas Mulawarman. The extraction of Mitragyna speciosa leaves using ChCl -Sor-based natural deep eutectic solvent and microwave-assisted extraction is detailed in Supplementary Data 3.

Chemical materials

The materials used in this study included choline chloride (ChCl; Xi’an Rongsheng Biotechnology, China), sorbitol (Sor; Chlorogreen, Indonesia), streptozotocin (STZ; Sigma-Aldrich, USA), penicillin spray (Jaya Supra Medica, Indonesia), filter paper, ketamine HCl injection (Dexa Medica, Indonesia), magnesium powder, distilled water (aquadest), glucose test strips (Nesco, Indonesia), and ethanol 96%. Additional reagents included polyvinyl alcohol, glycerine (Sigma-Aldrich, USA), alcohol swabs, Dragendorff’s reagent, hematoxylin and eosin (H&E) stain, ferric chloride (FeCl₃), and hydrochloric acid (HCl). Standard test animal feed, citrate buffer (pH 4.5), and 10% formalin buffer solution were also utilized in the experimental procedures.

Methods

Preparation of natural deep eutectic solvent

NADES composition used in this study was ChCl and Sor and prepared according to the previous study with some modifications.[20,21] Briefly, The ChCl-Sor mixture, with a molar ratio of 3:1, was heated to 80°C and stirred at 500 rpm until a clear solution was achieved. Subsequently, 35% v/v of distilled water was incorporated into the NADES.

Formulation of spray film-forming system

The formula used is listed in Table 1. The evaluation of the spray film-forming system was observed by organoleptic test (form, color, and odor), pH test, viscosity, and dry time. Extract concentrations were chosen based on a previous study conducted by de Moura et al.[22] and the orientation done prior to the formulation by assessing the color of the formula with various concentrations. In this study, the extract concentrations above 4% were found to be unfavorable due to their dark color.

Table 1.

Spray-film formulation

Materials	Percentage
Mitragyna speciosa extract	2	3	4
PVA	2.5	2.5	2.5
Aquadest	30	30	30
Glycerine	1	1	1
Ad ethanol 96	100	100	100

PVA: Polyvinyl alcohol

Procedures for establishing the type 1 diabetic wound animal model, which underpin the experimental design, are described in Supplementary Data 11–6.

RESULTS AND DISCUSSION

Formulation and evaluation of spray film-forming system

The formulated sprays have a watery form, dark orange color, and a characteristic odor of kratom extract [Supplementary Data 7]. The pH of sprays with 2%, 3%, and 4% NADES extract of kratom was 5.26 ± 0.09, 5.10 ± 0.15, and 4.88 ± 0.09, respectively. The pH between 4.5 and 6.5 is safe for the skin.[14] The viscosity evaluation of the spray film with 2%, 3%, and 4% of kratom extract resulted in viscosity values of 20.16 ± 1.692, 16.231 ± 0.827, and 16.782 ± 0.938 cPs, respectively. These water-like spray films were applicable for the skin. The dry time of the sprays was 2.37 min for the spray with 2% extract, 2.12 min for the spray with 3% extract, and 1.53 min for the spray with 4% extract. Film-forming sprays have numerous benefits over traditional topical formulations, including uniform medication distribution and dosage, enhanced bioavailability, reduced irritation, sustained drug release, and expedited wound healing via moisture regulation.[26]

The formulation, Blood glucose level groups, Histopathological rat, epithelial tissue, Neutrophil cell and epithelial tissue thickness scoring at those studies were prepared in three different concentrations, detailed in Supplementary Data 7–11.

Diabetic model

The blood glucose level of animal models before and after induction of STZ and day 14 after induction (within treatment period) was monitored to make sure that the rats were still in a diabetic condition [Supplementary Data 8]. STZ is utilized due to its strong affinity and selectivity for pancreatic beta cells in comparison to other diabetic agents.[27] The damage caused by STZ resembles that of type 1 DM, as it leads to reduced insulin production resulting from autoimmune damage to pancreatic beta cells, which in turn raises blood glucose levels. The experimental design and treatment grouping of animal models are described in Supplementary Data 4. The detailed protocol for constructing the diabetic wound model, including STZ induction and excisional wound creation, is provided in Supplementary Data 1.

The lifespan of mice and the research duration may not adequately reflect the chronic characteristics of diabetes wounds in humans since the use of STZ-induced mice elucidates early-stage wound healing deficiencies. A significant number of diabetic patients possess associated comorbidities that restrict effective wound healing. Although the STZ model does not completely mimic these settings, it offers a controlled setting to isolate the impact of hyperglycemia on wound healing.[28]

Wound healing activity

All rats were required to be in diabetic circumstances prior to treatment, except for the normal control group. Male Wistar rats were anesthetized with ketamine to alleviate pain after injury, as ketamine can induce muscle relaxation.[29] Table 2 shows the percentage of wound diameter of treated animal models during 16 days of observation. The percentage was calculated using Equitation 1.

Table 2.

Percentage of diabetic wound healing area

Groups	Percentage of wound diameter in rats (%)
	1	4	7	10	13	16
Positive Control	0.00±0.00	19.35±9.66	26.41±8.83	43.71±2.38	53.30±4.71	67.90±7.49
Negative Control	0.00±0.00	19.74±14.37	29.84±11.94	40.81±11.31	51.49±15.90	61.74±17.23
Normal Control	0.00±0.00	17.74±15.27	24.12±9.43	35.44±8.15	56.26±4.82	67.09±8.52
Extract Control	0.00±0.00	19.72±6.43	25.08±5.98	40.83±5.93	59.34±13.84	84.24±11.24
Spray-film 2%	0.00±0.00	30.96±9.04	40.68±16.95	58.30±14.80	71.77±1.05	81.49±2.18
Spray-film 3%	0.00±0.00	25.64±3.92	33.09±10.48	43.58±15.73	54.32±7.90	71.24±9.92
Spray-film 4%	0.00±0.00	30.91±5.19	44.82±6.82	59.72±7.23	71.70±5.89	83.99±12.02

Figure 1 presents that the percentage of wound healing in the spray-film 2%, 3%, and 4% groups was higher compared to the negative control group as well as that of the normal control group (P < α 0.05). The 4% Spray-film treatment exhibited notably superior outcomes in comparison to the positive control (P < α 0.05). In contrast, the 2% and 3% spray-film groups showed lower healing percentages compared to the positive control. The findings suggest that the 4% spray-film formulation demonstrated greater efficacy in enhancing wound healing. The increased concentration probably resulted in more potent therapeutic effects, facilitating more effective healing. However, the extract concentrations above 4% were found to be unfavorable due to its dark color and its viscosity. The method for wound diameter measurement and wound healing percentage calculation is described in Supplementary Data 5. Interestingly, the normal control experienced wound closure even though not faster than those of the treated group. The statistical analysis, including normality and homogeneity tests, is presented in Supplementary Data 6. This suggests that the body, under normal conditions, was able to restore itself home statically by repairing the damaged tissue and forming a new functional structure similar to its previous state. The application of kratom-based spray treatments in these groups likely supported or accelerated this natural healing process, improving tissue repair and regeneration.

Figure 1.

Percentage of wound diameter in rats (%)

The wound healing activity of the kratom spray film aligned with an in vitro, Zakaria et al., discovered that kratom extract enhances wound healing by facilitating rapid migration and angiogenesis in 3T3 fibroblast cells and HUVECs.[30] The primary processes contributing to the efficacy of natural products in wound healing therapies include antioxidant, anti-inflammatory, and antibacterial properties.[31] Many studies have revealed that phenolic compounds contained in kratom leaves are responsible for these properties. Zakaria et al.[32] reported that kratom leaf extract contains quinic acid, catechin, chlorogenic acid, caffeic acid, fraxin, neochlorogenic acid, quercetin, rutin, coumarin, and kaempferol. These compounds may have contributed to the wound healing activity in this study.

Figure 2 depicts the process of wound closure in diabetic rats observed every 3 days after initial treatment of the film spray for 16 days. Observation of appearance in wound healing reveals differences among the groups regarding the reduction in wound diameter. The distinctive wound heals as it presents itself, scabs appear on the skin, and epithelial cells start to develop.

Figure 2.

Wound healing process on diabetic rats

Skin histopathological observations

The quantity of neutrophil cells correlates with the intensity of inflammation; their levels will rise as the inflammatory state exacerbates. The nucleus of neutrophil cells has 2–4 lobulated segments and exhibits various geometries, including horseshoe and bean forms, as shown in Figure 3.

Figure 3.

Neutrophil cells in inflammatory phase rat skin

Neutrophil cells are abundant during the inflammatory phase of wound healing, which typically occurs within the first 24 h after skin damage and can last up to 2 weeks.[33] The primary role of this phase is to remove dead tissue, clear the inflamed area, and eliminate foreign bodies and bacteria. While neutrophils are crucial for the initial stages of wound healing, their prolonged presence beyond 2 weeks can hinder the healing process. The preparation of skin samples and hematoxylin and eosin staining procedures was conducted as outlined in Supplementary Data 2. Extended inflammation, characterized by sustained neutrophil activity, can delay the transition to the next healing phases, such as tissue repair and remodeling, ultimately slowing down the overall wound healing process.[34] Remodeling is the subsequent phase following the inflammatory phase. Remodeling is the process by which epidermal cells move from the edge of the wound to the wound area and cover the wound, consequently closing the wound.[35] The formation of the epidermis is related to the parameters of wound success, because the faster the formation of the epidermis, the faster the wound healing.

Supplementary Data 9 indicates that neutrophil cell scoring for all groups was high on day 14, which aligns with the typical inflammatory response in wounded tissue. Neutrophil cells on the rat skin tissue (H and E staining) of the control and treatment groups are presented in Supplementary Data 10. The presence of neutrophils indicates active inflammation. As the first line of innate immunity, neutrophils perform phagocytosis to eliminate pathogens and clear necrotic tissue, thereby preventing the spread of infection. By day 21, a reduction in neutrophil cell scoring was noted. This suggests that the spray treatment groups exert anti-inflammatory effects on diabetic wounds, helping to reduce neutrophil accumulation. The decrease in neutrophil cells is attributed to the active flavonoids and alkaloids in kratom, which are believed to inhibit inflammatory mediators, thereby preventing excessive neutrophil accumulation and promoting a more balanced inflammatory response.[36] During the later stages of the inflammatory phase, neutrophils are either engulfed by macrophages or experience apoptosis (programmed cell death) as the wound moves toward the resolution phase. The decrease in neutrophil cells indicates a shift to the subsequent stage of healing, during which the inflammatory response starts to diminish, and the tissue maturation process continues.[33] However, the mechanisms employed by neutrophils to eliminate potential pathogens can also inflict considerable tissue damage on the host. This additional damage may result in prolonged recovery and extensive scar development. Most of the host damage inflicted by neutrophils is attributable to the activity of proteases released by these cells. The clinical significance of this issue is shown by multiple studies indicating that elevated levels of neutrophil-derived proteases correlate with chronic, nonhealing wounds.[37] The wound closure in the animal models in this study more likely indicates the potential of the polyphenol-rich extract formulated in the spray film to inhibit the protease’s activity. Thus, more research is needed to assess the potential of the film spray on neutralizing neutrophil proteases to enhance chronic wound healing while maintaining neutrophils’ ability to kill pathogens and lower the risk of infection.

In this study, epidermal thickness was assessed on day 14, with the thickness continuing to increase until day 21. The spray treatment groups demonstrated significantly thicker epithelial tissue on day 21 compared to the negative control. Histopathological observations [Supplementary Data 9] of the spray treatment group on day 21 revealed a well-formed and relatively thick epidermis in contrast to the negative control. This effect is likely due to the active flavonoid compounds in kratom, which promote the migration and proliferation of epithelial cells, thus accelerating the formation of the epidermis. The formation of a thick epidermis is crucial in wound healing, as it helps close the wound more quickly. These findings suggest that kratom can aid in wound healing by reducing neutrophil numbers and enhancing epidermal thickness, thereby supporting the healing process. The effectiveness of kratom spray as a wound healer is comparable to that of positive control [Supplementary Data 11]. Spray formula with 4% concentration has the best effect on decreasing neutrophil cells and increasing epidermis formation.

CONCLUSIONS

All the developed spray films exhibited appropriate pH, viscosity, and organoleptic characteristics, rendering them suitable for application on the skin. Among the tested formulations, the one containing 4% of the kratom leaf NADES extract showed enhanced wound healing activity on diabetic rats highlighting its potential as an effective therapeutic option for managing diabetic wounds. The spray film-forming system formulation of M. speciosa NADES extracts demonstrated notable healing effects on diabetic wound by reducing wound diameter, decreasing neutrophil cells, and increasing epithelial tissue thickness.

Conflicts of interest

There are no conflicts of interest.

SUPPLEMENTARY DATA: Supplementary Data 1:

Construction of animal models of type 1 diabetic wounds

Diabetic model protocol: Wistar strain male rats aged about 3 months (150–200 g) were acclimatized in cage for 1 week prior to the induction of streptozotocin (STZ). The rats were fed with growers’ feed and water. The cage was illuminated with 12-h light/12-h dark cycle in laboratory condition (temperature 22°C ± 2°C, humidity 60%–70%).[23]

Prior to STZ induction, the rats underwent an overnight fast. Blood samples were collected for the initial measurement of glucose levels before induction. The rats received an injection of STZ intraperitoneally at a dosage of 60 mg/kg BW. The blood glucose levels were measured 72 h after injection, as STZ has a relatively long half-life, necessitating time for glucose levels to rise.

Incisional wound model: Wounding was conducted once the blood sugar level of the test animals exceeded 200 mg/dL. An excision wound measuring 2 cm in diameter on the back of the rat, located 5 cm from the ear and with a depth of 0.3 cm, parallel to the vertebrae. The skin was subsequently preserved in neutral buffered formalin solution for additional processing in the preparation of histopathological samples.

Supplementary Data 2:

Skin histopathological observations

The injured area of the skin was carefully removed and subsequently inserted and preserved in a 10% neutral buffered formalin solution, followed by hematoxylin and eosin staining for histopathological examination. The samples were observed under a microscope with ×400.

Supplementary Data 3:

The extraction of Mitragyna speciosa leaves

Microwave-assisted extraction (MAE) using choline chloride–sorbitol (ChCl-Sor) as a solvent was performed to produce polyphenol-rich extracts from kratom leaves. One hundred grams of powdered kratom leaves were placed into a round-bottom flask. Subsequently, 1000 mL of ChCl-Sor was added to the mixture, which was then subjected to MAE. The microwave settings were set as follows: irradiation time of 20 minutes, microwave power of 60% W, and a solid–liquid ratio of 1:10. Subsequently, the natural deep eutectic solvent extract of kratom leaves was separated by filtration. The obtained extract was subsequently stored in sealed vials and protected from light exposure.

Supplementary Data 4:

Experiment design

All experiments were performed in accordance with the protocol approved by the Research Ethical Committees of Universitas Ahmad Dahlan (ethical approval under number 012406120). The wounded rats were divided into seven groups, each consisting of five rats. These groups included normal, negative control, positive control, kratom natural deep eutectic solvent NADES extract, spray film with concentration of 2%, 3%, and 4% kratom NADES extracts. The positive and negative groups were treated with penicillin spray and film-spray base without extract, respectively. Spray film treatments were applied to each rat twice daily in the morning and afternoon. Construction of animal models of type 1 diabetic wounds is shown in Supplementary Data 1.

Supplementary Data 5:

Wound healing analysis

Measurement of wound diameter: The wound diameter of the rats was measured according to the Morton method[24] and performed every 3 days. Observations were conducted from day 1 to day 16 following the creation of the wounds. The percentage of diabetic wound healing area was calculated using Equation 1.

Where Px is the percentage of wound healing on day x (%), d1 is 1^st-day wound diameter (cm), and dx is wound diameter on day x (cm).

Skin histopathological observations: To investigate the polymorphonuclear cell numbers in the wound area and epithelial tissue thickness, the rat was sacrificed following the procedure in Supplementary Data 2.

Supplementary Data 6:

Statistical analysis

Measuring involved the diameter of the healed wound utilizing the Shapiro–Wilk method for normality testing and the Levene test for homogeneity assessment.[25] To assess the variations in wound healing efficacy among test animals utilizing the Kruskal–Wallis and Mann–Whitney tests.

Supplementary Data 7:

Spray film-forming system of Mitragyna speciosa leaf natural deep eutectic solvent extract: (a) 2% of extract, (b) 3% of extract, (c) 4% of extract

Supplementary Data 8:

Blood glucose level (mg/dL)
Groups	Before STZ induction	After STZ induction	Day 14
Positive control	104±10.41	264.75±86.97	416.75±127.51
Negative control	122.5±12.53	341.75±137.42	470.75±143.29
Normal control	148.67±62.08	Not induced	134.50±20.27
Extract control	126.75±9.25	285.75±20.46	278.50±25.83
Spray film 2%	127.75±5.76	290±86.21	320.00±64.20
Spray film 3%	117.75±10.40	336±96.51	401.25±132.71
Spray film 4%	131.25±10.05	271±138.4	366.50±86.12

STZ: Streptozotocin

Supplementary Data 9:

Neutrophil cells on histopathological rat skin tissue at ×400

Supplementary Data 10:

Epithelial tissue thickness of rat skin tissue

Supplementary Data 11:

Neutrophil cell and epithelial tissue thickness scoring
Groups	Scoring
	Neutrophil cella		Epithelial tissue thicknessb
	Day 14	Day 21	Day 14	Day 21
Positive control	++++	+	+	++++
Negative control	++++	+++	+	+
Normal control	++++	++	+	+++
Extract control	++	+	+	+++
Spray-film 2%	++++	+	+	++
Spray-film 3%	++++	++	++	+++
Spray-film 4%	+++	+	++	+++

^aIntensity of neutrophil cell: “+” indicates very low, “++” indicates low, “+++” indicates high, “++++” indicates very high, ^bEpithelial tissue thickness: “+” indicates thinner, “++” indicates less thick, “+++” indicates thicker, “++++” indicates thickest

Funding Statement

Kemdiktisaintek No. 049/PFR/LPPM UAD/VI/2024