INTRODUCTION:

Diabetes mellitus (DM) is a disease that is frightening in society because it causes every 10 seconds in the world of people¹. DM is also identified with symptoms of hyperglycemia caused by defects in insulin secretion, insulin action and metabolic disorders². WHO provides an instruction that all herbal medicines must be scientifically evaluated for their activity³. There are many formulations to treat diabetes mellitus but scientific validation is lacking³. Treatment of insulin resistance by increasing antioxidant activity can increase glucose uptake by hemidiaphragm rats⁴. Wound healing is a complex and dynamic process because it is a bio-cellular and biochemical unit that occurs continuously.

The wound healing process consists of several phases, namely the coagulation, inflammation, proliferation, and remodeling phases⁵. Wound healing is also defined as a complex and dynamic process that results in improvement of the anatomical structure and function of the tissue⁵. Wounds in diabetic patients involve disorders of the peripheral and autonomic nerves and greatly, glucose affects the wound healing process of diabetes mellitus^5,6.

One of the advances in medicine in the field of science and technology is the emergence of nanotechnology. Treatment of various serious diseases without side effects can be treated with nanomaterials and nanostructures⁷. The advantages of nanotechnology are reducing toxic, economical, and environmentally friendly⁷. The nanostructures of lipid carriers are colloid carrier systems that have many advantages such as increased bioavailability, increased solubility and therapeutic efficacy². The nanostructured lipid carrier formulation is very effective in the treatment of diabetic wound healing². Plant-based nanoparticles are receiving increasing attention because of their non-toxic, cost-effective, and relatively simple⁸. Herbal plants can act as a promising antidiabetic agent in streptozotocin-induced diabetic rats⁹. The role of medicinal plants in various biomedical applications has been proven. Plant extracts have been tested for their antidiabetic activity against streptozotocin-induced toxicity in male albino rats¹⁰. The application of natural medicine has been widely applied empirically by the community in the treatment of diabetes¹. The conversion of herbs to nanoparticles is a target for drug penetration in the skin and has an enhanced skin targeting effect¹¹.

IL-1β activity is required for healing. Levels of IL-1β, which is known to be a major key in inflammation¹². The role of increasing cell apoptosis and suppressing cell proliferation is the mechanism underlying the postponement in wound healing. Diabetes is known to cause tissue damage by inducing apoptosis in endothelial cells with increased expression of IL-1β¹². During the diabetic wound healing process, IL-1β is upregulated in diabetic macrophages in mice, whereas diabetes increases IL-1β expression in macrophages mediated by oxidative stress-induced inflammatory activation¹³. Hyperglycemia-induced oxidative stress in monocytes also inhibits the VEGFR1 signaling pathways^12,13. Increased oxidative stress and suppressing VEGFR1 signaling in macrophages constitutively activate other intracellular signaling pathways and lead to IL-1β production¹³.

The medicinal plant Rhodomyrtus tomentosa (Aiton), also known as haramonting, is widespread in Indonesia. The leaves are used to treat diarrhea, indigestion, bleeding, and acute gastroenteritis¹⁴. Nano herbal haramonting can also improve the histology of the placental organs, liver and kidney preeclampsia^15,16. Nano herbal haramonting can also affect testicular histology, abundance, kidneys, liver and lungs exposed to cigarette smoke^17-22. Recent research from Rhodomyrtus tomentosa has seven phloroglucino derivatives that have cytotoxic activity on HeLa cells and anti-inflammatory activity¹⁴.

The medicinal plant Zantoxylum acanthopodium, also known as andaliman, is also spread in Indonesia. The fruits have the high antioxidants to increase HSP-70 expression and reduce Malondialdehyde (MDA)²³. Andaliman nano herbal has low toxicity and is safe in several organs so that it can be developed into medicine²⁴. Andaliman nano herbal can also repair the liver, kidney, and placenta in preeclamptic rats^23,25,26. The aim of the study is diabetic wound healing by haramonting leaves and andaliman fruits in diabetic rats.

MATERIAL AND METHODS:

Preparation of Zanthoxylum acanthopodium and Rhodomyrtus tomentosa:

Andaliman fruit is taken from andaliman fruit traders from Dairi, North Sumatera, and the fresh green of haramonting leaves are obtained from Gunung Tua District, North Padang Lawas Regency. Haramonting and Andaliman are used as nano herbal preparations. Haramonting herbal nano preparations in the form of Self Nano-Emulsifying Drug Delivery System (SNEDDS) using High Energy Milling (HEM) at Indonesian research institutes (LIPI, Jakarta)^24,27.

Animal Handling:

This study used 100 healthy and fertile rats (Rattus norvegicus) aged 8-11 weeks weighing 150-200 g. The rats were obtained from the Medan, North Sumatera, Animal Disease Investigation Center. Rats were fed and drank adlibitum. Rats cages are kept clean and set 12 hours light - 12 hours dark. Handling of experimental animals in accordance with the requirements of the applicable code of conduct. Before the research was conducted, an application was submitted to get ethical clearance to the Health Research Ethics Commission for the North Sumatera Region of Medan.

Study design:

The research type is true experiment with the randomized posttest only control group research design which was conducted in the laboratory. This study consisted of 4 groups for each treatment; K1: negative control, K2: MEBO, K3: Nano herbal of andaliman fruits (100mg/kg BW), and K4: Nano herbal of haramonting leaves (100mg/kgBW). The treatments were observed on days 0, 4, 8, 12, and the 16th. Each group had 5 replications on every treatment day so that each group needed 20 rats. Diabetic model rats with alloxan Injection (120mg/kg.IP) in all groups intra-peritoneally in the abdominal area (stomach) and rats were declared diabetes mellitus when the blood glucose levels reached ≥ 200mg/dL. Measurement of blood glucose levels was carried out from the tail blood vessels, namely the lateral veins using a glucometer glucoDR Bio-sensor. The MEBO (Moist Exposed Burn Ointment) Bioplasenton is also given to experimental diabetic wounds as a comparison.²⁸.

The experiment of diabetic wound rats:

The hair of diabetic rats was shaved and then cleaned with alcohol in the lateral area of the back about 3 cm from below the ear. The rats were injected with Ketamine-HCl (dose 50mg/kg BW) intramuscularly in the femur area as an anesthetic to reduce pain. Making of diabetic wounds using an iron plate measuring 1.5cm x 1 cm. The iron plate was heated with blue fire until the iron was red for 3 minutes then the iron plate was placed on the skin of the mouse's back for 2 seconds to make second-degree burns. The experimental animals were dissected by cervical dislocation on the 4th, 8th, 12th, and 16th days.

Hematoxylin Eosin Staining:

The skin tissue was performed on the 0th, 4th, 8th, 12th and 16th days. The skin tissue was cut vertically in size of 1.5cm x 1.5cm to the hypodermis layer of skin. Then, the tissue was placed in an organ tube and fixed with a 10% BNF (Neutral Formaline Buffer) solution for 24 hours. Dehydration was done by immersing in alcohol with a grade of 30%, 50%, 70%, 85%, 95% and 2 times absolute alcohol for 30 minutes each. Clearing is done using alcohol and xylol. Tissue staining was performed by giving Hematoxylin for 15 seconds, and Eosin staining for 15-20 minutes. Observation at 400x magnification.

Immunohistochemistry staining of IL-1β:

IL-1β detection used a RPA563Ra01. Recombinat Interleukin 1 Beta (IL-1β) poliklonal mouse,. The tissue was cut using a microtome with a thickness of 4-6 microns. For pre-treatment, the tissue was heated in citrate buffer at pH 6.0 and 350 W. After washing with PBS, the tissue was incubated with IL-1β antibodies, respectively, at 37°C then washed again with PBS before applying avidin–biotin peroxidase. Lastly, all sections were visualized using a chromogen DAB working solution followed by counterstained with Meyer’s hematoxylin.

Data analysis:

Research data using SPSS software version 23 using the Anova test at 5% level then continued with the Post Hoc-Duncan test. If the data are not normally distributed and/or the variance is not homogeneous, then we use the Kruskal Wallis test and then proceed with the Mann-whiney test.

RESULTS:

Fig.1: Histology of rats skins tissue before and after treatment for 16 days. K1: negative control, K2: MEBO, K3: Nano herbal of andaliman fruits (100mg/kg BW), and K4: Nano herbal of haramonting leaves (100mg/kg BW). (0) day of first treatment, (4) fourth day, (8) eighth day, (12) twelfth day, (16) sixteenth day.

Wound healing was seen on day 16 in the treatment of nano herbal haramonting and andaliman (Fig. 1). Histologically in healing, the wound is characterized by a thick, granular vascular tissue dominated by extensive fibroblasts and collagen. The epithelium has mostly covered the wound on the skin. Epithelial-filled skin tissue indicates the presence of epithelial cell proliferation. Likewise, many fibroblasts and collagen have formed. This proves the existence of cell proliferation that occurs in the injured skin layer. Figure 1 showed that some of the compounds contained in the nano herbal of haramonting and andaliman can stimulate cell division and growth to form scar tissue such as flavonoids, steroids, saponins, and tannins.

Fig. 2. Expression of IL-1β after various treatments and wound healing time of rats skin tissue before and after treatment for 16 days. K1: negative control, K2: MEBO, K3: Nano herbal of andaliman fruits (100mg/kg BW), and K4: Nano herbal of haramonting leaves (100mg/kg BW). (0) day of first treatment, (4) fourth day, (8) eighth day, (12) twelfth day, (16) sixteenth day.

Table 1: Mean Rank of IL-1β expressions

Groups	Days
Groups	0	4	8	12	16
K1	17.00	41.80	66.30	93.70	80.40
K2	11.00	23.40	33.60	80.20	56.40
K3	11.70	23.40	17.90	51.20	86.80
K4	14.00	38.40	66.90	84.60	58.80

K1: negative control, K2: MEBO, K3: Nano herbal of andaliman fruits (100mg/kg BW), and K4: Nano herbal of haramonting leaves (100mg/kg BW)

The administration of MEBO and nano herbal of andaliman and haramonting had the same effect in diabetic wound healing in rats skin. There were significant differences between all groups (P <0.001) in IL-1β expressions. It showed that there was an increase from the first day until 16 days in diabetic wound healing (Table 1 and Fig. 3). The release of the cytokine IL-1β indicates a proinflammatory process that is triggered by wounds. Wounds cause excessive production of pro-inflammatory cytokines such as IL-1β which results in prolongation of the inflammatory phase so that the wound will take a long time to heal. So that compounds/substances are needed that can act as immunoregulators in balancing excess production of the cytokine IL-1β and can stimulate the formation of new tissue in the skin. Herbal medicines that can play a role in balancing the excess production of the cytokine IL-1β and stimulate the formation of new tissue in the skin are haramonting and andaliman.

Fig. 3. Boxplot of IL-1β expression after various nano herbal treatments and wound healing time. K1: negative control, K2: MEBO, K3: Nano herbal of andaliman fruits (100mg/kg BW), and K4: Nano herbal of haramonting leaves (100mg/kg BW). (0) day of first treatment, (4) fourth day, (8) eighth day, (12) twelfth day, (16) sixteenth day.

DISCUSSION:

Wounds can result in loss of the skin's defense barrier, allowing bacteria or fungi to penetrate deeper into the tissues and blood vessels, putting them at risk of systemic infection. The inflammatory process in the wound healing process plays a role in fighting infection at the beginning of the wound, where in the first 24 hours the neutrophil cells, lymphocytes and macrophages fight infection, clean the cellular matrix debris and foreign substances¹².Furthermore, neutrophils secrete pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 to degrade the remaining cellular matrix. Wounds cause the production of pro-inflammatory cytokines such as IL-1β excess which results in prolongation of the inflammatory phase so that the wound will take a long time to heal¹³. So, the compounds/substances that can act as immunoregulators are needed in balancing the excess production of the cytokine IL-1β and can stimulate the formation of new tissue in the skin^12,13. The administration of MEBO and the administration of andaliman and haramonting nano herbs had the same effect in wound healing in diabetic rats.

Diabetes conditions cause metabolic disorders to the body so that it interferes with the proliferation of skin cells. On the time of wouds the skin tissue will trigger an increase in Ca²⁺intacellule and the formation of H₂O₂ or an increase in extracellular ATP. This ATP release is known as DAMPs (Damage associated molecular patterns)^12,29. Then binds to the P2Y receptor purine on healthy cells, releasing a cytoplasmic signal that causes activation of intracellular Ca²⁺ and metalloproteinases¹². This process results in the release of an EGFR ligand, namely HB-EGF (Heparin-binding EGF-like growth factor)^12,30.

The release of ATP and Ca²⁺ can activate 4 pathways, namely: (1) cpG DNA with TLR 9, (2) Introduction of ligands from bacteria, namely lipopolysaccharide (LPS) known by TLR4. (3) Flagella by TLR 5 (3), and (4) Flagella by NLRP3 in the cytosol. On lines (1), (2) and (3) will activate MyD88^12,31. TIR domain of MyD88 interacts with TIR domain of TLR to activate 2 serine threonin protein kinase IRAK4 (IL1-receptor associated kinase 4) and IRAK 1^31,32. The IRAK complex recruits TNF receptor associated factor 6 (TRAF 6), an E3 ubicuitin ligase. E2 ligase of TRICA1 acts together with TRAF 6 to produce a polyubiquitin chain scaffold on TRAF 6 itself and activates serine threonine kinase TAK 1 (Transforming growth factor beta-avtivated kinase 1) and MAP (Mitogen-activated protein) kinase¹². TAK 1 perforates and activates the IκB kinase (IKK) complex composed of 3 proteins: IKKα, IKKβ and IKKγ or known as NEMO^29-32. Activation of IKK will phosphorylate IκB and then degrade and release NF-κB. NF-κB then enters the nucleus and activates proinflammatory cytokines, especially IL-1β²⁹. Then in line (4) there is a bond between Flagella by NLRP3^12,34. This bond will form an oligomer and bind to a protein adapter called ASC. Then the bond between the ASC and the sensor from NLRP3 will activate incaspase-1, the bacterial material in the form of a flagellum (rod) will enter the cytosol via T3SS and activate pro-IL1β1^2,13,29. Pro-IL1β will interact with incaspase-1 to activate IL-1β. In addition to PI3K-AKT and ERK-MAPK signals on epithelial cells. In addition, the wounds area will experience hypoxia, this hypoxic state will stimulate fibroblast activation^12,34.

Wound healing is a natural process through the collective movement of epithelial cells which involves three processes, namely inflammation, proliferation, and maturation³⁵. Many herbs are formulated to treat diabetes mellitus but validated scientific and medical data are lacking³⁶. A herbal has natural active compounds, its nutritional substances are useful for the care and treatment of wounds and other human health³⁷. Diabetes mellitus is also caused by malfunctioning of the pancreas which is responsible for the production of the hormone insulin and inflammatory mediators^38,39. Healing of diabetic wounds by administering ointments such as poly-herbal ointment had a significant wound healing effect in diabetic rats⁴⁰. Musa paradisiaca L ointment has also been shown to have a hemostatic effect that can stop bleeding and has the potential to heal wounds⁴¹. The gel and juice of Musa paradisiaca was also evaluated for their wound healing activity^42,43. Preparation of drugs with nanoparticles for wound healing has been done with solid lipid nanoparticles (SLN) consisting of a dense matrix⁴³. Nanoparticulate drug delivery represents a promising drug delivery system⁴⁴.

Increased glucose levels in people with diabetes mellitus will be able to affect angiogenesis which triggers the wound to heal. Normal angiogenesis depends on a balance between blood vessel growth, proliferation, and maturation of blood vessels⁴⁵. Diabetes mellitus can significantly disrupt this balancing process by disrupting healing, tissue regeneration, and restoring the normal vascular system^45,46. Endothelial cells that are exposed to glucose for a long period of time are dysfunctional and prone to apoptosis⁴⁶.

Flavonoids are antioxidants that play a role in protecting ascorbic acid from reactive oxygen species (ROS) as an ingredient needed for collagen synthesis by inhibiting cyclooxygenase and lipoxygenase, thereby limiting the number of inflammatory cells migrating to wound tissue⁴⁷. It causes an inflammatory reaction and the exposure time to the wound tissue with ROS is shorter.

The active substances of tannins and saponins act as antioxidants and antimicrobials, decreased wound contraction, and accelerate epithelialization⁴⁸. The content of flavonoids is also found in andaliman fruits and haramonting leaves The proliferation of fibroblasts and collagen has been formed more in the administration of nano herbal andaliman and haramonting. This is evidenced in the histology of Figures 1 and 2 where there is cell proliferation that occurs in the injured skin layer. It shows that some of the compounds contained in the nano herbal of haramonting and andaliman stimulate cell division and growth to form wound tissue such as flavonoids, steroids, saponins, and tannins⁴⁹. The histology of skin tissue is also almost the same as that of MEBO administration. Therefore, Nano herbal andaliman and haramonting can be developed into herbs that can be used to treat wounds in diabetes.

CONCLUSION:

There was a significant difference between all groups (P <0.001) in IL-1β expression in rat's skin tissue. Nano herbal of haramonting and andaliman can stimulate cell division and growth to new form skin tissue in diabetic rats' wounds..

ACKNOWLEDGEMENT:

This study acknowledges grant support from the Deputy for Strengthening Research and Development, Ministry of Research and Technology/ National Research and Innovation Agency in Doctoral Dissertation Reserach grant Number:28/UN.5.2.3.1/PPM/KP-DRPP/ 2020.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 26.11.2020 Modified on 08.06.2021

Research J. Pharm. and Tech. 2022; 15(5):2041-2046.

DOI: 10.52711/0974-360X.2022.00337