Antiinflammatory Effect of Andrographolide in Sambiloto Extract ( Andrographis paniculata) on Ulcerative Colitis

—Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the gastrointestinal tract which is one of the inflammatory bowel diseases (IBD). In Indonesia, epidemiological data obtained from hospital reports, generally show that the incidence of UC is higher than Crohn's Disease (CD). Mesalamine as a drug of choice for UC, is related to some side effects. Therefore, herbal plants such as sambiloto (Andrographis paniculata) could be used as a complementary therapy in UC. The purpose of this article is to provide information about the potential mechanisms of andrographolide (AG) as a bioactive compound in sambiloto (Andrographis paniculata) extract as an anti-inflammatory agent. The method used by authors in this article is a narrative review method, by collecting studies about the anti-inflammatory effect of AG on UC through a database search. The results showed that one of the ingredients of sambiloto, diterpenoid labdane compound in the active form of AG, is able to inhibit the expression of pro-inflammatory cytokines so that it has the potential to act as an anti-inflammatory similar to mesalamine in UC therapy. Additionally, sambiloto contains flavonoids and polyphenols which serve as antioxidants. In conclusion, AG has an anti-inflammatory property that might be utilized as a part of UC complementary therapy.


INTRODUCTION
Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the gastrointestinal tract which is one of the inflammatory bowel diseases (IBD) [1].Patients typically present with bloody diarrhea, with a genetic predisposition after environmental exposures are thought to develop ulcerative colitis, abnormalities in the gut epithelial barrier, the microbiota, and a dysregulated immune response are significantly associated with the development of ulcerative colitis.The diagnosis is made using a combination of clinical, biological, endoscopic, and histological evidence [2].
Worldwide, North America and Northern Europe have the highest incidence and prevalence of inflammatory bowel disorders globally.There is a strong correlation between a Westernized lifestyle and environment and inflammatory bowel disease.The annual incidence of ulcerative colitis is 9-20 cases per 100,000 people.Its annual prevalence ranges from 156 to 291 cases per 100,000 people.Adults are more likely to have ulcerative colitis than Crohn's disease, which is more common in pediatric patients [3].Epidemiological data shows that UC, which was formerly thought to occur more commonly in industrialized nations, is now really on the rise in underdeveloped and developed nations alike including those in Asia, such as Indonesia, over the last two decades [4].
The pathophysiology of ulcerative colitis involves defects in the immune system, leukocyte recruitment, epithelial barrier, and colon microbiota [3].Some experts suggest that IBD is an autoimmune disorder in which the immune system of the host attacks the cells and tissues of the gastrointestinal tract (GIT).These inflammatory manifestations include two diseases, namely: Crohn's disease (CD) and UC.UC is characterized by a lifelong condition that causes inflammation of the colonic mucosa, starting in the rectum, and extending proximally.Common symptoms that often appear are bloody diarrhea, abdominal cramping, urgency, tenesmus, and fever.Most people with UC experience periods when they experience symptoms (flare-ups or exacerbation), followed by longer periods of no symptoms (remission) [5,6].Administering 5-aminosalicylates, antibiotics, steroids and immunomodulators has been used to manage ulcerative colitis and reduce symptoms and their remissions.However, longterm use of these drugs can result in mild to severe toxicity as well as the possibility of UCrelated complications.Even with the use of related drugs, about 15% of patients even have to undergo colectomy or proctocolectomy, despite taking related drugs [7].
The aim of medical management is, first, to induce a rapid clinical response and normalize biomarkers and, second, to maintain clinical remission and reach endoscopic normalization to prevent long-term disability.Corticosteroids and medications containing 5aminosalicylic acid such as mesalamine, are used as remission-inducing therapies [2].Mesalamine is well known for its main side effect: renal toxicity so that monitoring renal function before and during the use of this drug is required.Other conditions such as: bone marrow suppression should also be monitored with CBC, particularly in older individuals.There is also a chance of liver failure, cholestatic hepatitis, liver insufficiency, and other hepatotoxic signs and symptoms.The liver function needs to be monitored for long-term use of mesalamine [8,9].With such serious complications, inefficiency for some patients, and high recurrence rates, thus investigating a novel, safe, and cost-effective treatment strategy for UC is needed.
Alternative therapies based on phytopharmaceuticals have been researched to be a part of the strategy for preventing complications as well as medications used over an extended period of time, with minimal side effects.These therapies can be used as adjuvant therapy or first-line therapy in patients who are less responsive or unresponsive to mesalamine [10].The purpose of this paper is to review the therapeutic effects of andrographolide (AG) from sambiloto extract that are potentially beneficial to human health particularly regarding the pathogenesis of UC.Several have been demonstrated by several in vitro and in vivo studies have demonstrated these effects, which relate to the underlying mechanism of UC and provide additional insight about AG in sambiloto extract as a newer modality, of natural, and safe complementary medicine for UC treatment in the future.

METHOD
This literature review was carried out using the narrative review method.The literature search used several keywords such as: "Andrographis paniculata", "ulcerative colitis", "andrographolide" "anti-inflammatory", and "inflammatory bowel disease".Literature search was carried out using Google Scholar, PubMed, and Science Direct search engines.
The inclusion criteria consist of: (1) original articles or review articles published in the last 10 years, (2) full text available in English or Indonesian language, (3) The studies discussed the antiinflammatory effect of AG on UC.The exclusion criteria consist of: (1) full text not available, (2) the study is written in other than English or Indonesian language.
After obtaining the studies that matched inclusion criteria, the studies were assessed and compiled according to titles, study designs, methods and doses, results, and conclusions.Mechanisms of AG as anti-inflammatory against oxidative stress in UC are summarized and discussed in this literature review.

RESULT
We found 6 articles that matched our inclusion criteria, and we presented the summary of those studies in Table 1 below.

Pathology of Ulcerative Colitis
By microscopic findings, the GIT consists of four layers, each of which reflects its anatomical and physiological functions, namely: mucosa, submucosa, tunica muscularis externa, and tunica adventitia [11].The inner lining of the digestive tract, or mucosa, is crucial in creating the mucus barrier or mucus between internal organs and the digestive tract lumen.This layer, which is mostly made up of epithelial cells joined by tight junctions, is crucial for the transfer of nutrients as well as for blocking the entry of pathogenic microbes and substances that are harmful to the host [12].In the mucosal layer, goblet cells and endocrine cells establish the epithelium.Endocrine cells work to secrete specific hormones that assist in digestion, while goblet cells are the primary cell type of the crypts in the left side of the colon, producng mucin that forms water-soluble mucus.This unique structure of the colon with parallel test tube-like arrangement of crypts differentiated by a consistent amount of lamina propria in between, can display chronic injury when there is an expansion of this lamina propria and a loss of crypts [13].
In UC, there is a defect in the synthesis and secretion of mucus in the mucosal layer, creating damage to the integrity of the tight junctions in the epithelial layer and causing increased mucosal permeability making it easier for antigens to enter from both environmental exposures, toxins, and pathogenic microorganisms.The mucosa and submucosa are both affected by the progression of the inflammatory process [6].There is a sharp border between normal and affected tissue.The mucosal membrane is erythematous, granular, and friable, with loss of the typical vascular pattern and often followed by scattered hemorrhagic areas.Only in severe manifestations which involving the tunica muscularis, distinguished by large mucosal ulcers and abundant purulent discharge [14].Antigen exposure to the immune system in the mucosa activates macrophages and dendritic cells which will present antigens and triggers a series of inflammatory responses, triggering the production of proinflammatory cytokines and differentiation of T-cells into effector T-helper (Th) cells.Th cells are part of the lymphocytes which mediate the immune response.Th also activates other immune cells in determining the specificity of antibodies secreted by B cells [15].After proliferating, Th differentiates into: Th1, Th2, and several other derivatives.Th1 initiates the cell-mediated immune response by releasing proinflammatory cytokines like interferon gamma (IFN-), tumor necrosis factor alpha (TNF-), and interleukin-2 (IL-2).Whereas, Th2 stimulates the humoral immune response by producing anti-inflammatory cytokines like: IL-4, IL-5, IL-6, and IL-10.Th1 cytokines stimulate the development of Th1 cells to suppress Th2 cells in a reciprocal interconnection between Th1 and Th2 lymphocytes.Th2 cytokines, on the other hand, cause Th2 cells to suppress Th1 cells.So that under normal conditions there is homeostasis between the number of Th2 and Th1 cells.The increased polarization of Th1 is believed to be closely related to the pathogenesis and chronic inflammatory response in UC [15,16].
Other immune responses from dendritic cells also trigger Toll-like receptors (TLRs) which initiate the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) which stimulates the production of other pro-inflammatory cytokines such as: IL-12, IL-23, and IL-1β , along with mitogen-activated protein kinase (MAPK) and their upstream signaling pathways, induce the AMP activated protein kinase (AMPK) pathway in LPS-induced macrophages which also contributes to chronic inflammatory responses.These conditions facilitate the recruitment of leukocytes to the GIT mucosa which is mediated by the release of chemoattractants such as CXCL8 which is found in the incidence of UC and amplifies the inflammatory response [17,18].

Mechanism of AG as Anti-inflammatory & Potential Antioxidant Agent for Ulcerative Colitis
] is a labdane diterpenoid that has been isolated from the stem and leaves of A. paniculata or sambiloto and has been studied for its therapeutic effects including anti-inflammatory, anti-platelet aggregation, antioxidant, immunomodulation, and potential antineoplastic properties.The extract is presented by colorless bicyclic diterpene lactone crystals and is extremely bitter.AG is contained in all sections of the sambiloto, but the highest levels are found in the leaves [19].Sambiloto also has several other active compounds such as: deoxyandrographolide, diterpene glycosides, lactones, flavonoids, flavonoid glycosides, and polyphenols.Some other literature explains that the leaves and roots of the plant as a whole have also been used as antiinflammation and anti-bacteria herbal medicine since ancient times for various diseases in Asia and Europe [20].
Inflammation is the main pathophysiology underlying the course of UC disease.A study by Fragoulis et al (2016) explained that the IL-23/IL-17 axis pathway in CD4+ T lymphocytes also plays important role in the expression of proinflammatory factors such as: TNF-α, IL-1β, and IL-6, which are closely associated with the pathogenesis of UC, and serve as the foundation for the novel development of anti-inflammatory therapeutic for UC.AG inhibits TNF-α expression thereby suppressing TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) and also inhibits TNF-induced monocyte and endothelial adhesion, which are both key factors in the inflammatory responses and progression in the colonic mucosa [21,22].Another subsequent study by Zhu et al (2018) demonstrated that AG extract inhibited Th17 activity in CD4+ cells found in the colonic mucosa of mice with TNBS-induced colitis (4,6-trinitrobenzenesulfonic acid) morphology, thereby inhibiting the production of IL-23, IL-17, and ROR-Ɣ (Th17 transcription factor) at several concentrations (0, 10, 20 and 30 µg/mL) in combination with mesalamine.
Additionally, peripheral blood mononuclear cells (PBMCs) were isolated from UC patients, AG demonstrated inhibitory effects on Th17 cell percentages, proinflammatory factor levels, and relative protein expression at several concentrations as stated previously, resulting in the relief of UC [23].These findings were consistent with a study related to the treatment of IL-23/IL-17 axis-mediated diseases such as UC, Crohn's disease, psoriasis and spondyloarthritis, which showed that AG performed similar efficacy to mesalamine.The data suggested that AG inhibits Th1/Th17 response while increasing the concentrations of IL-4 (a Th2 cell produced cytokine) in the culture medium, the percentages of Th2 cells, and the protein levels of GATA-3 (Th2 lineage-specific transcription factor), resulting in the balance of Th1/Th17/Th2 as part of the UC management.However, whether andrographolide affects the T cell responses of UC patients has not been explored [10].
In addition, previous studies by Guo et al (2014) showed the anti-inflammatory effect of AG by inhibiting NLRP-3 inflammasome in macrophages.AG reduced in a concentration-dependent inhibition of pro-inflammatory IL1B secretion from lipopolysaccharide (LPS)-treated human monocytic THP-1 cells and murine bone marrow-derived macrophages (BMDM) by the ELISA assay.Furthermore, the activation of CASP1 (as indicated by the presence of the cleaved form and enzyme activity) was also significantly inhibited by AG, thus protecting the mucosal layer and lamina propria in conjunction with active colitis which correlated with the severity of inflammation, and tumor development [24,25].Other anti-inflammatory effects were also shown by AG by inhibiting proinflammatory cytokines including NF-κB and MAPK in colon tissue, upstream the signaling pathways, as well as activating the AMPK pathway in LPSinduced macrophages, thus attenuating DSS-induced intestinal barrier dysfunction and inflammation [17].AG also inhibits the expression of proinflammatory cytokines including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), which are involved in the synthesis of prostaglandin E2 (PGE2) and free radical molecules nitric oxide (NO), by preventing Nf-kB from binding to host cell DNA [26], while it showed pro-resolving function by accelerating M1 to M2 macrophage conversion and up-regulating resolution-related genes (IL-10, TGF-β, and HO-1) [27].
AG also showed its potential antioxidant activity by preventing the activation and binding of iNOS to lipopolysaccharide (LPS) in the mucosal cell walls, so that the formation of NO molecules can be suppressed.Additionally, the phytochemical analysis of the extracts revealed variability in the peak of the non-standardized constituents, which are part of the flavonoid and phenylcarboxylic acid.It is widely known that flavonoids and phenolic acids work well as antioxidants and free radical scavengers, while oxidative stress is known to contribute to inflammatory tissue damage and plays a role in cytokine signaling including in UC as well [28,29].Several other in vitro studies have also reported that AG has an anti-inflammatory effect by inhibiting macrophage and neutrophil adhesion-transmigration mediated by macrophage adhesion molecule-1 (Mac-1), thus contributing in suppressing the production of reactive oxygen species (ROS) mediated by protein kinase C (PKC).In addition, AG reduced the formation of the proteins caspase 3/8 and Bax as well as the phosphorylation of p38 in a Balb/c mouse model of intestinal mucositis caused by 5-fluorouracil (5-Fu) [26,30].

CONCLUSION
UC is an idiopathic chronic inflammatory disease of the GIT that occurs due to exposure to antigens originating from food, environmental factors and microorganisms that invade the mucosal lining of the GIT due to tight junction damage of the mucosal barrier.UC is commonly seen with symptoms like bloody diarrhea, abdominal pain, nausea and vomiting.One of the many medicinal plants easily found in Indonesia is sambiloto (Andrographis paniculata) which has been extensively researched and developed for both the characteristics and the pharmacological effects of the bioactive compound andrographolide (AG) which has numerous therapeutic benefits.AG is widely used as an anti-inflammatory agent with potential effects as an that is believed to help improve the colonic mucosa by reducing and preventing the inflammatory process that occurs in the pathogenesis of UC, thus helping to control and decrease the severity of its symptoms, progression, recurrence, and UC-related complications as well as reducing unwanted side effects from the long-term drug use.
Further and advanced preclinical studies are needed in various colitis model trials followed by clinical studies to explore the efficacy and biological effects of AG and other active compounds contained in sambiloto in several range of doses as well as development in various preparations and techniques especially in Indonesia, with newer and modern extraction methods, to obtain optimal efficacy without triggering toxicity.AG performed antiinflammatory/ pro-resolving effects, therefore relieving UC effectively.(Zhou et al., 2023)

Figure 1 .
Figure 1.Mechanism effect of andrographolide (AG) extract on the pathogenesis of UC.