A Comparative Study on The Anti-inflammatory Activity of Boerhavia Diffusa and Scoparia Dulcis

1.1 Inflammation

Inflammation is a complex biological reaction of vascular tissues to adverse stimuli such as infections, damaged cells, and irritants. It occurs when body tissues are exposed to infections and physiological tissue damage ^[1]. Inflammation is a vital physiological response that are important in deciding whether tissues survive or are destroyed following a variety of traumas. As a defence mechanism, inflammation has developed in higher species in reaction to harmful stimuli such tissue damage, microbial infection, and other unpleasant circumstances. It's a vital host immunological response that permits the elimination of dangerous stimuli and the repair of injured tissue.  This defence response involves immune cells, changes to blood vessels, and molecular mediators. Inflammation is represented by the development of granulomas, leukocyte infiltration, and edema.Endogenous stress signals, also known as structural pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), are the cause of inflammation, according to the receptors for pattern recognition (PRRs) encoded in the germline and this PRRs are expressed by various kinds of cells, but it is mainly ex pressed on myeloid cells, including monocytes, macrophages, neutrophils, and dendritic cells. It is crucial to note that during inflammation, each tissue displays unique inflammatory features due to both local and general molecular, immunological, and physiological factors.  It is now abundantly evident that inflammation has both beneficial and detrimental effects, acting as two sides of a single sword. Understanding the regulating mechanisms and their boundaries will aid in the future comprehension and treatment of a variety of illnesses. Developing conceptual and mechanistic knowledge of inflammation leads to novel therapeutic methods and insights into human diseases. ^[2]

1.1.1 Types of inflammation

 Inflammation is mainly classified into two types, acute inflammation and chronic inflammation. Acute inflammation is a physiological process that occurs swiftly in response to injury or infection, typically manifesting within minutes to hours and lasting for a short duration, often resolving within days. This rapid onset is marked by the exudation of plasma proteins and fluids from the bloodstream into the surrounding tissue, leading to localized swelling and redness. A hallmark of acute inflammation is the migration of leukocytes, especially neutrophils, to the site of injury. In addition to combating infections, the acute inflammatory response also serves to facilitate tissue repair. The damaged body part and personal characteristics determine the cause and length of the condition. By recruiting additional immune cells and releasing signalling molecules, this process helps to clear out debris and promote healing in the affected area ^[3]. When inflammatory processes continue after they are needed or happen without a genuine stimulation, it is known as chronic inflammation. Chronic inflammation frequently results from Infections, abnormal immune reactions, obesity, exposure to environmental triggers, unhealthy diet, smoking, sleep problems, high stress levels and that can lead to the consequences like DNA damage, cancer, atherosclerosis, heart disease, Alzheimer's disease, asthma, rheumatoid arthritis, diabetes, and other conditions ^[4].

1.1.2 Causes of Inflammation

 Various physical and chemical agents can cause inflammation. The physical agents are mechanical injuries, extreme temperatures, radiation, triggering inflammatory pathways and the chemical agents that leads to the inflammation are a growing array of drugs and environmental toxins. Biological agents like bacteria, viruses, fungi, and parasites play a significant role in the development of inflammation ^[5]. Additionally, immunologic disorders, characterized by hypersensitivity reactions, autoimmunity, and immunodeficiency states, can lead to inappropriate or insufficient inflammatory responses. Genetic and metabolic disorders, such as gout and diabetes mellitus, often involve inflammatory mechanisms as part of their pathophysiology, highlighting the multifaceted nature of inflammation as both a protective and a pathological process in the body ^[6].

1.1.3 Pathophysiology

Inflammation is a complex immune response triggered by injury, infection, or harmful stimuli, involving vascular changes, immune cell activation, and the release of signalling molecules. The development of inflammation involves a series of coordinated steps that aim to eliminate harmful stimuli, repair tissue damage, nnaand restore homeostasis ^[7]. The process begins with the release of pro-inflammatory mediators like cytokines, histamines, and prostaglandins. A key component of the inflammatory response is the COX (cyclooxygenase) pathway, where the enzyme COX-1 and COX-2 convert arachidonic acid into prostaglandins and thromboxane. COX-1 is constitutively expressed and maintains normal physiological functions, such as protecting the stomach lining, while COX-2 is induced during inflammation and produces prostaglandins that promote pain, fever, and vasodilation. These prostaglandins increase vascular permeability, allowing immune cells and proteins to reach the site of injury or infection. Chronic inflammation can lead to tissue damage and fibrosis, often associated with dysregulated COX activity, contributing to various inflammatory diseases, such as arthritis ^[8].

1.1.4 Current Treatment

Three main categories of medications used to treat inflammatory reactions.
Corticosteroids, DMARDS, or disease-modifying anti-rheumatoid medications NSAIDs, or non-steroidal anti-inflammatory drugs, include naproxen, ibuprofen, and aspirin. Many inflammatory illnesses can be effectively treated with these commonly used medications. For the treatment of inflammation, NSAIDs are most frequently recommended ^[9]. In order to minimize temperature and pain during inflammation, NSAIDs work by inhibiting the activity of COX which is implicated in inhibiting PG production. The NSAIDs are known for their three primary effects: analgesia (pain reduction), anti-inflammatory, and antipyretic (fever reduction). They provide an anti-inflammatory effect that helps treat a number of ailments, such as rheumatoid arthritis, osteoarthritis, musculoskeletal diseases, and pericarditis. Analgesia is used to relieve mild to severe pain. They don't lead to dependence, but their maximum therapeutic efficacy is far less than that of opioids. By inhibiting the COX enzymes and lowering prostaglandins throughout the body, nonsteroidal anti-inflammatory medications (NSAIDs) lower fever, discomfort, and inflammation. NSAIDs are frequently used to treat both acute and chronic disease symptoms. Asthma, psoriasis, and arthritis are examples of chronic inflammatory conditions that have been treated with NSAIDs ^[10].

1.2 Herbal Remedies & its significance

 India is one of the largest storehouses of medicinal plants in the world, and herbal remedies are very common in India. Herbal remedies are used for various ailments due to their immediate availability, accessibility, and affordability. About 50% of modern drugs are derived from plants, and plant products have pharmacological properties including inflammatory, antipyretic, and analgesic activities ^[11]. Plants have the ability to synthesize a wide variety of phytochemical compounds, and consumption of plant products reduces the risk of developing pathological conditions such as cancer, nervous system disorders, cardiovascular, genetic, and inflammatory diseases. One major drawback of the powerful anti-inflammatory synthetic medications currently on the market is their toxicity and the recurrence of symptoms after stopping use.  Long-term NSAID use damages the cardiovascular and renal systems and causes gastrointestinal ulcers can cause a number of serious adverse effects, including osteoporosis, diabetes mellitus, insulin resistance, hyperglycaemia, and anxiety.   Non-selective NSAIDs can cause serious injury to the upper gastrointestinal tract, including perforations, ulcers, and bleeding. COX-2 selective inhibitors are associated with less upper gastrointestinal tract toxicity ^[12]. These adverse effects can be reduced by the use of herbal drugs having ant inflammatory activity Many researchers have isolated a greater number of phytocompounds from various medicinal plants and reported as anti-inflammatory agents. These phytocompounds inhibit the anti-inflammatory mediators. The main target was Cyclooxygenase (COX) enzyme. Large number of herbal species has been used traditionally or as folk medicines against inflammatory disorders. Many of them have been studied scientifically and proved to be beneficial anti-inflammatory agents. Despite the divergent bioactivities of the plant medicines against various diseases, active components of most plant extracts have not been elucidated thoroughly, due their complex mixtures ^[13]. However, the core chemical classes of anti-inflammatory agents from natural sources have been reported to engage a vast range of compounds such as polyphenols, flavonoids, terpenoids, alkaloids, anthraquinones, lignans, polysaccharides, saponins and peptides. Some plants with anti-inflammatory activity are listed below ^[14].

Taxonomy

Botanical name: Boerhavia diffusa

Family: Nyctaginaceae

Order: Caryophylales

Genus: Boerhavia                                                

Phylum: Tracheophyta

Kingdom: Plantae

Class: Magnoliopsida

Species: Boerhavia diffusa L.

Figure 1. Boerhavia Diffusa

Boerhavia diffusa, a creeping perennial weed commonly found in tropical and subtropical regions, is a well-known ethno-medicinal plant. Various parts of the plant, including the leaves, roots, and stems, as well as its extracts, have been extensively used in traditional and folk medicine to treat a wide range of ailments. The plant contains a diverse array of phytochemicals, such as flavonoids (e.g., C-methyl flavone, 5,7-dihydroxy-3’,4’-dimethoxy-6,8-dimethylflavone, borhavone), alkaloids (like punarnavine), glycosides (such as punarnavoside and eupalitin), rotenoids (boeravinones A-H), as well as steroids, triterpenoids, lipids, lignans, carbohydrates, proteins, and glycoproteins ^[15]. Numerous studies have confirmed the biological, pharmacological, and clinical activities of the plant and its constituents. Notable therapeutic effects include diuretic, hepatoprotective, anti-inflammatory, anti-cancer, anti-diabetic, and immuno-modulatory properties, as well as anti-fibrinolytic, anti-lymphoproliferative, and analgesic effects. It is also used in the treatment of pulmonary tuberculosis. In addition, the plant has shown some less prominent activities, including non-teratogenic, antioxidant, anti-viral effects against plant viruses, anti-bacterial, anti-fungal, adaptogenic, anti-amoebic, lipotropic, and anticonvulsant activities. Because of these proved pharmacological actions this plant has a major role in the medical world ^[16].

1.4 Scoparia Dulcis & its Significance

Taxonomy

Botanical name: Scoparia dulcis Linn.

Family: Scrophulariaceae

Kingdom: Plantae

Subkingdom: Trachcobionta

Division: Magnoliophyta

Genus: Scoparia

Class: Magnoliopsida

Subclass: Asteridae                                       

Order:  Lamiales                                       

Phylum: Tracheophyta

Species: Dulcis

Figure 2. Scoparia dulcis

Scoparia dulcis commonly known as ‘sweet broom weed’ is distributed throughout the tropical and subtropical region of the world. The plant is used traditionally as used as a remedy for treating various disease. Its ethno-medicinal uses amongst various indigenous tribes in the rain-forest zone are well-documented. In fresh or dried form S. dulcis plants have been traditionally used as remedies for Diabetes mellitus in India and hypertension in Taiwan. It is used in curing ailments such as fever, diarrhoea, ulcer, cancer, wounds, skin rash, cough and tuberculosis. The fresh or dried plant has been used for treating stomach aches, inflammation, bronchitis, hemorrhoids and hepatosis. In the western part of Orissa its root is traditionally is used as an effective remedy for Jaundice and diarrhoea. It is also used as an analgesic and antipyretic, in stomach troubles, bronchitis, as well as inhibition of herpes simplex virus replication, gastric H+,K+-ATPase activation and antitumor activity. It is deemed to be a panacea for all ills. In Gambia, a lotion prepared from the plant is used in curing fever. A hot water infusion or decoction of the leaves or whole plant is used medicinally by indigenous tribes of Nicaragua to treat malaria, stomach disorders, menstrual disorders, insect bites, fevers, heart problems, liver disorders and venereal diseases. It has been used for blood cleansing, in childbirth and as a general tonic^{[16 c]}.  Scoparia dulcis is a rich source of various bioactive compounds, including flavones, terpenes, steroids, phenols, tannins, saponins, amino acids, coumarins, and carbohydrates. Key chemical constituents of the plant are scopadulcic acids A and B, scopadiol, scopadulciol, scopadulin, scoparic acids A–C, and betulinic acid. Additionally, other notable compounds present are acacetin, amyrin, apigenin, benzoxazin, benzoxazolin, benzoxazolinone, cirsimarin, cirsitakaoside, coixol, coumaric acid, cynaroside, daucosterol, dulcinol, dulcioic acid, gentisic acid, glutinol, hymenoxin, linarin, luteolin, mannitol, scoparinol, scutellarein, scutellarin, sitosterol, stigmasterol, taraxerol, vicenin, and vitexin which are responsible for the wide range of pharmacological actions ^[17]. Traditionally the fresh or dried plant has been used as a remedy for treating diseases such as, stomach ailments, kidney stones, hypertension, diabetes, inflammation, bronchitis, haemorrhoids, analgesic, antipyretic and urinary disorders. Plant is also used for upper respiratory bacterial and viral infections, to relieve from all types of pain, to tone balance, strengthen heart function, for veneral diseases and urinary tract infections. The leaf of Scoparia dulcis is used for diabetes in India. Plant is also reported to possess cytotoxic, anti-cancerous, antimicrobial, anti-malarial, anti-ulcer, antacid, ant cholesterol and antioxidant actions ^[18].

1.5 Problem in Hand & Solution

Existing anti-inflammatory drugs, such as NSAIDs and corticosteroids, have several notable deficiencies. They often come with significant side effects, including gastrointestinal issues, cardiovascular risks, and potential kidney damage. Many patients experience limited efficacy, requiring higher doses that can exacerbate side effects. Additionally, these medications may have a short duration of action, necessitating frequent dosing, and some, like opioids, pose a risk of dependence. Corticosteroids can suppress the immune system, increasing infection risk, while newer biologics may be prohibitively expensive. The complexity of their use, potential for drug interactions, and delayed onset of action further complicate their effectiveness, underscoring the need for better therapeutic options. Herbal remedies often have fewer side effects, making them suitable for long-term use without the risk of dependency or severe adverse reactions ^[19]. The use of plants as therapeutic agents serves multiple objectives, including the isolation and concentration of bioactive compounds for direct use as drugs, the production of novel or known bioactive substances for semi-synthesis into more potent, patentable entities with improved efficacy or reduced toxicity, and the use of plant compounds as pharmacological tools for research. Additionally, whole plants or their parts may be utilized as herbal remedies in traditional medicine. A key distinction of plant-derived drugs is their molecular diversity, which far exceeds that of synthetic compounds, despite advancements in chemical synthesis. This diversity provides a broader range of biological functions and potential therapeutic applications, making plant-derived compounds promising candidates for developing treatments for various diseases. The complexity and variability of these natural products provides opportunities for discovering novel drugs with unique mechanisms of action that may offer advantages over conventional synthetic medications. ^[20]

2. Aim &Objectives

2.1. Aim

To conduct a study on the anti-inflammatory activity of Scoparia dulcis and Boerhavia diffusa and compare it.

2.2 Objectives

1. Identify and collect the plants Scoparia dulcis and Boerhavia diffusa.

2.Prepare the alcoholic extracts of the plants Scoparia dulcis and Boerhavia diffusa.

3. Evaluate the anti-inflammatory activity of Scoparia dulcis and Boerhavia diffusa.

4.Compare the anti-inflammatory activity of Scoparia dulcis and Boerhavia diffusa

3. MATERIALS AND METHODS

3.1. Materials Used

3.2.1 Collection of Scoparia dulcis

For the present study, the investigated plant Scoparia dulcis were collected from Ettumanoor, Kottayam district on October 24^th 2024. The collected plant parts (complete herb) were sorted to remove any undesirable materials or plant sections. They were dried in shade for two week and powdered to a coarse form.

Figure 3. Scoparia Dulcis plant

Figure 4. Dried powder of scoparia Dulcis

3.2.2 Collection of Boerhavia Diffusa

The whole plant of Boerhavia diffusa were collected from Pala, Kottayam district on 1/11/2024. They were dried in shade for three weeks and ground to a coarse form.

Figure 5: Boerhavia Diffusa Plant

Figure 6. Dried powder of Boerhavia diffusa

3.3 Preparation of Extract

3.3.1 Extraction of Scoparia Dulcis

Macerate the dry powder of Scoparia dulcis in ethanol at a ratio of 1:4. Kept the mixture at room temperature with occasional stirring for 5 days. Then, concentrated and dried the filtrate using a rotary vacuum evaporator [46].

Figure 7. Extraction of Scoparia Dulcis

Figure 8. Extract of Scoparia Dulcis

3.3.2 Extraction of Boerhavia Diffusa

100 grams of the dried powder of Boerhavia Diffusa is taken into a sterile iodine flask,500 millilitres of ethanol is added and shaked vigorously. Covered the bottle tightly and let it sit for 5 days. Filtered the mixture with Whatman filter paper at room temperature. Concentrated the extract, dried it at room temperature, and stored it in a refrigerator [47].

Figure 9. Extraction of Boerhavia Diffusa

Figure10.Extract of Boerhavia Diffusa

3.3 Calculation of Percentage Yield

The percentage yield was calculated for each plant extract and major compounds with reference to the crude material taken using the formula. Percentage yield with reference to crude plant material = (Weight in grams of extract obtained /Weight in grams of plant material taken) x 100

3.4 In vitro anti-inflammatory screening methods

3.4.1 Protein Denaturation Using Egg Albumin

Preparation of 1% of egg albumin solution:

For making egg-albumin solution using a fresh hen’s egg properly involves carefully cracking an egg, transferring 1 mL of the translucent portion to 100 mL of w/V distilled water, and stirring thoroughly. The clear component of the egg is called egg albumin. The water should be cold when making the solution. Water will coagulate if it is heated to a boil.

Procedure:

Unknown crude extracts' anti-inflammatory properties can be assessed in vitro by preventing the denaturation of the protein egg albumin. To create a reaction mixture with a total volume of 5 mL, 0.2 mL of 1-2% egg albumin solution (from fresh hen's eggs or commercially available egg albumin powder), 2 mL of sample extract or standard (Diclofenac sodium), and 2.8 mL of phosphate buffered saline (pH 7.4) were combined. 2.8 mL of phosphate-buffered saline, 0.2 mL of 1-2% egg albumin solution, and 2 mL of distilled water were combined to make a total volume of 5 mL of the control. After 30 minutes of incubation at 37±2°C, the reaction mixtures will be heated for 15 minutes in a water bath at 70±2°C. After cooling, distilled water was used as the blank and the absorbance was measured at 280 nm using an appropriate UV/Vis spectrophotometer ^[48].

3.4.2 Human red blood cell membrane stabilization method

The in vitro HRBC (Human Red Blood Cell) membrane stabilization method is used for evaluating anti-inflammatory activity of plants. Fresh blood is collected and mixed with an equal volume of sterile saline to prepare a suspension of red blood cells (RBCs). 1 ml of phosphate buffer, 2 ml hyposaline and 0.5 ml of HRBC suspension were added to the prepared plant extracts. It was incubated at 37 °C for 30 min and centrifuged at 3000 rpm for 20 min. Absorbance of the released haemoglobin is measured using a spectrophotometer at 560nm. Diclofenac sodium was used as reference standard and the percentage inhibition of haemolysis is calculated. ^[49,50]

3.8   Statistical Analysis

Values are expressed as mean ± Standard error mean, where n = 6. The results were analysed by one-way analysis of variance [ ANOVA] followed by post Dunnett's post hoc multiple comparison test. The difference between groups were considered significant at P<0.05*, P<0.01**, P,0.001***, P<0.0001****. The statistical analysis was carried out using Graph Pad Prism version 10.4.1 for windows was used to analyze the data

4. RESULTS AND DISCUSSION

4.1. RESULTS

4.1.1Practical Yield of the Plant Extracts

The plant extracts were collected and the practical yield was calculated for each plant.

4.1.2. Anti-inflammatory activity by HRBC membrane stabilization

Anti-inflammatory activity of Scoparia dulcis and Boerhavia diffusa in HRBC membrane stabilization was evaluated by measuring percentage inhibition. (Table.3)

All the values are in Mean±SEM. ^*P<0.05,   ^**P<0.01,  ^***P<0.001,   ^****P<0.0001 when compared to control.   ^#P<0.05,  ^##P<0.01,  ^###P<0.001 when compared to standard.

Figure11.  Anti-inflammatory activity of Boerhavia diffusa by HRBC Method

Figure 12. Anti-inflammatory activity of Scoparia dulcis by HRBC Method

4.1.4 Anti-inflammatory activity by protein denaturation method

Protein denaturation using egg albumin is an effective in vitro anti-inflammatory screening technique to evaluate the effect of test drugs by measuring percentage inhibition of protein denaturation. (Table.4)

All the values are in Mean±SEM. ^*P<0.05,   ^**P<0.01,  ^***P<0.001,   ^****P<0.0001 when compared to control.   ^#P<0.05,  ^##P<0.01,  ^###P<0.001 when compared to standard.

Figure 13. Anti-inflammatory activity of Boerhavia diffusa by Protein denaturation

Figure 14. Anti-inflammatory activity of Scoparia dulcis by Protein denaturation