New Potential and Characterization of Andrographis echioides L. Ness Stem Extract as an Anti-Arthritic Agent

1. Introduction

Plants have been used in synthetic drug development and non-pharmacological applications due to their medicinal components. Traditional medicine has used herbal remedies to combat fungal and insect threats since prehistoric times [1]. Despite the widespread use of medicinal plants, their value remains unrecognized in many countries. Phytochemicals, found in plants, play a crucial role in human health by reducing platelet aggregation, activating detoxifying enzymes, exerting antioxidant effects, stimulating the immune system, and demonstrating antibacterial properties [2]. These compounds, including alkaloids, carotenoids, saponins, tannins, phenols, steroids, and flavonoids, have disease prevention capabilities, including antiparasitic, anticancer, antidiabetic, antiaging, antidepressant, and antimicrobial effects [3]. They also contribute to wound healing, accumulate bioactive substances in fruits and vegetables, and enhance stress tolerance [4]. Flavonoids are predominant in plants, with many fruits and vegetables containing anticarcinogenic varieties [5]. Anthocyanins, carotenoids, and lycopene are natural antioxidants found in various fruits and vegetables.

The therapeutic use of natural products, especially those derived from plants, has seen a resurgence in popularity in recent years. These products, depending on their specific attributes, can be categorized as pharmaceuticals, cosmetics, medical devices, nutritional supplements, or traditional foods [6]. Over the past two decades, a significant number of newly approved chemical entities have been small-molecule natural products (SMNPs) or SMNP-based compounds [7]. As a result, governments, international organizations, and businesses are actively exploring medicinal herbs and natural products as a valuable source for new drug compounds. This renewed interest in plant derivatives is partly due to the increased adverse effects associated with conventional medicines. However, plant-based products face several challenges, such as an unpleasant taste and strong odor, which can negatively impact patient compliance. Additionally, their limited solubility, slow dissolution rate, and instability at high pH levels restrict their therapeutic application, often requiring large dosages to achieve the desired effect [8]. Moreover, rapid oxidation and degradation during storage can reduce the concentration of active ingredients in plant derivatives, thereby diminishing their nutritional value.

Rheumatoid arthritis is an inflammation of the joints, primarily caused by a weak autoimmune system and swelling. Symptoms include weight loss, low fever, poor appetite, feeling unwell, and irritation around joints. The disease is not congenital and can be caused by environmental influences or infection [9]. Autoimmune diseases, such as lumps, atherosclerosis, and cancer, can be treated with antioxidants and a healthy diet. Citrus fruits, which contain essential nutrients like carotenoids, selenium, folic acid, potassium, and vitamin C, can also help protect against human malignancies [10]. Arthritis is a chronic condition that causes irregular function, inflammation, and pain. There are 100 types of arthritis, including rheumatoid arthritis, osteoarthritis, and infectious arthritis. Rheumatoid arthritis results from the immune system attacking joints, while osteoarthritis occurs when cartilage becomes tired or less movable [11]. Infectious arthritis is caused by bacteria and viruses, and antibiotics are used to cure it. Synovitis, a type of arthritis, is directly linked to rheumatoid arthritis. Treatment depends on the type and suitability of the individual, with the initial step being a general examination by a doctor. Herbal and nutritional supplements have become important in therapeutic practice and research for rheumatology and orthopedics [12].

Andrographis echioides is an herbaceous plant found in dry areas of southern Asian countries, with a flowering season from March to June and October to December. The plant has elongated leaves with glandular hairs, a slightly quadrangular stem, and a raceme-type inflorescence [13]. The flower’s calyx is lanceolate with glandular hairs, and the capsules are ovoid, sparsely hairy, pointed above, and narrowed below. The plant has an average number of capsules per plant and four seeds per capsule. The plant’s anatomy includes a well-defined epidermis with epidermal hairs, a well-defined hypodermis, and a chlorenchymatous cortex. The root is composed of a single layer of barrel-shaped epidermal cells, lacking stomata and cuticle, and a compactly arranged parenchymatous cortex [14]. Andrographis echioides has been found to have various pharmacological activities. It has been found to have diuretic activity, antimicrobial activity, anthelmintic activity, hepatoprotective and antioxidant effects, anti-inflammatory, analgesic, and antipyretic activity, and anti-ulcer activity. The plant’s ethanol extract has shown potential anti-ulcer activity in all tested models [15]. Additionally, the plant’s leaves have been subjected to Soxhlet extraction using ethanol as a solvent, showing potential for controlling A. aegypti larvae. These findings suggest that Andrographis echioides may be a valuable tool for treating various diseases and infections. Further research is needed to isolate the active principles responsible for these promising properties.

2. Materials and methods

3. Results

3.1 Phytochemical analysis

The examination delineates the active biomolecules identified within the stem extract. In light of this finding, the stem of A. echioides has been deemed a focal point for the biosynthesis of bioactive compounds. The identified compounds include terpenoids, flavonoids, steroids, alkaloids, saponins, tannins, cardiac glucosides, phlorotannins, proteins, coumarin, emodin, anthraquinone, anthocyanin, carbohydrates, leucoanthocyanin, phenols, alkaloids, and xanthoproteins. Among these constituents, with the exception of leucoanthocyanins and phlobatannins, all others exhibited the anticipated results (Figure 1, Table 1). The quantitative assessment of flavonoids, tannins, saponins, phenols, alkaloids, and terpenoids indicates potential outcomes. Notably, with the exception of tannins, the remaining phytonutrients, particularly saponins, displayed significant results (Figure 2, Table 2).

S.No	Phytochemical constituents	Andrographis echioides
1	Terpenoids	+++
2	Flavanoids	+++
3	Saponin	+++
4	Tannin	+++
5	Alkaloids	+++
6	Steroids	+++
7	Glycosides	+++
8	Phlobotannins	—
9	Protein	+++
10	Coumarin	+++
11	Emodin	++
12	Anthroquinone	+++
13	Anthocyanin	+++
14	Carbohydrate	++
15	Leucoanthocyanin	—
16	Cardiac glycosides	+++
17	Xanthoproteins	+++
18	Phenol	++

Table 1.

Qualitative analysis of the ethanolic extract of Andrographis echioides stem.

(+ = slightly present, ++ = moderately present, +++ = strongly present, − = absence)

S.No	Phytochemical constituents	Empty Value (mg/g)	Yield value (mg/g)	Final Value (mg/g)
1.	Phenol	18.830	18.846	0.016
2.	Flavonoid	20.656	20.669	0.013
3.	Tannin	20.286	20.326	0.04
4.	Saponin	21.046	21.088	0.42
5.	Alkaloids	19.325	19.595	0.27
6.	Terpenoids	18.771	18.785	0.014

Table 2.

Quantitative analysis of ethanolic extract of Andrographis echioides.

3.2 Protein denaturation model

The results show that Andrographis echioides stem extract inhibits protein denaturation in a dose-dependent manner, similar to diclofenac sodium. At 100 μg/mL, the extract (0.37%) is slightly less effective than diclofenac (0.27%), indicating notable anti-inflammatory potential (Figures 3 and 4, Table 3). The findings suggest that Andrographis echioides may serve as a natural anti-arthritic agent, supporting its traditional medicine use.

S.No	Concentration	Protein denaturation (%)
		Andrographis echioides	Diclofenac sodium
1	20 (μg/ml)	0.68	0.55
2	40 (μg/ml)	0.54	0.47
3	60(μg/ml)	0.50	0.39
4	80 (μg /ml)	0.43	0.33
5	100 (μg /ml)	0.37	0.27

Table 3.

Protein denaturation assay of Andrographis echioides stem.

3.3 Anti-arthritic activity

The HRBC membrane stabilization assay demonstrates a dose-dependent anti-inflammatory effect of Andrographis echioides, comparable to diclofenac sodium. As the concentration increases, the membrane stabilization percentage improves, indicating its ability to prevent hemolysis and protect cell membranes from damage. At 100 μg/mL, the extract shows 0.22% stabilization, while diclofenac sodium exhibits 0.50%, with an overall 81.3% stabilization effect (Figures 5 and 6, Table 4). Although slightly less effective than diclofenac, Andrographis echioides still exhibits significant anti-arthritic potential, supporting its traditional use in inflammation management and suggesting its role as a natural alternative for arthritis treatment.

SI.No	Concentrations	HRBC Membrane Stabilization Assay (%)
		Andgraphis echioides	Diclofenac sodium	Percentage %
1	20(μg /ml)	0.58	0.72	50.8%
2	40(μg /ml)	0.51	0.65	56.7%
3	60(μg/ml)	0.44	0.62	62.7%
4	80(μg/ml)	0.38	0.55	67.7%
5	100(μg/ml)	0.22	0.50	81.3%

Table 4.

In vitro anti-arthritic activity of the stem of Andrographis echioides using HRBC.

4. Discussion

Research conducted by Gurupriya and Cathrine in 2022 revealed that the stem of Andrographis echoides contains a variety of biologically active compounds, including steroids, terpenoids, saponins, tannins, alkaloids, and flavonoids. These compounds exhibit efficacy against microorganisms, liver toxins, inflammation, tumors, and free radicals [24]. An investigation focused on the phytochemical composition of Ocimum species ethanolic extract. Their analysis identified the presence of phenols, glycosides, flavonoids, terpenoids, and saponins in the extract [25]. Likely, further studies by Pavithra Kumari corroborated the presence of various phytochemicals in Ocimum species, specifically phenols, flavonoids, saponins, tannins, and alkaloids. Notably, this species exhibited a particularly high concentration of saponins compared to other phytochemicals [26].

A study reported on the quantitative analysis of moisture content, including volatile components, in the tested drug. Their findings emphasized the importance of the final drying process and the rate of moisture removal from secondary metabolites in determining the drug’s therapeutic efficacy. Additionally, they highlighted the significance of saponin compounds for isolating diosgenin and their potential in anti-arthritic applications [27].

Barleria lupulina has demonstrated anti-inflammatory activity through the inhibition of protein denaturation. An ethanolic leaf extract of the plant was tested, and the results showed inhibition percentages of 53.78%, 55.25%, and 60.6% at different concentrations in the protein denaturation test. Diclofenac sodium, used as a standard, showed inhibition percentages of 63.3%, 75%, and 79%. The study finally suggested that B. lupulina has potential as a remedy for oxidative stress [28].

Another study evaluated the anti-inflammatory activity of ethanol extract and fractions of B. sapida stem bark using in vitro methods, specifically albumin denaturation and membrane stabilization assays. The results showed that the extract and fractions exhibited anti-inflammatory activity by inhibiting heat-induced albumin denaturation and stabilizing red blood cell membranes at concentrations of 200–1000 μg/mL and 50–250 μg/mL, respectively. The ethyl acetate fraction, which had the highest phenolic and flavonoid content, was a key contributor to this activity [29].

The study investigates the anti-inflammatory and anti-arthritic activities of Martynia annua ethanolic fruit extract. The extract was extracted using ethanol and phytochemically investigated. The results showed antioxidant activity of 49.1 and anti-hyaluronidase activity of 84.60, indicating its potential for anti-arthritic and anti-inflammatory properties. The extract’s potential is significant for treating inflammation and rheumatoid arthritis [30]. Another study assessed the anti-arthritic and anti-inflammatory activities of the methanolic extract of Peganum harmala leaves in vitro and in vivo. The extract showed significant antioxidant, anti-inflammatory, and anti-arthritic activities primarily due to alkaloids, flavonoids, and phenols. It also attenuated polyarthritis, weight loss, anemia, and paw edema in polyarthritic rats. The results suggest that P. harmala extract has potential therapeutic benefits [31].

5. Summary and conclusion

This investigation demonstrates that the phytochemical screening of the ethanolic extract of A. echioides revealed an abundant presence of terpenoids, flavonoids, saponins, tannins, alkaloids, steroids, glycosides, proteins, coumarins, anthraquinones, anthocyanins, cardial glycosides, and xanthoproteins. Phenols, carbohydrates, and emodine were detected in moderate quantities, while phlobatannins and leucoanthocyanins were not observed. Quantitative assessment of the ethanolic extract indicated high saponin content (0.042 mg/g), low tannin levels (0.04 mg/g), and intermediate concentrations of alkaloids (0.27 mg/g), terpenoids (0.014 mg/g), and flavonoids (0.13 mg/g). The ethanolic extract of A. echioides demonstrated inhibition of protein (egg albumin) denaturation at concentrations ranging from 20 to 100 μg/ml. Diclofenac sodium, utilized as a reference drug, stabilized the HRBC membrane across the same concentration range. Similarly, the ethanolic stem extract of A. echioides exhibited HRBC membrane stabilization at equivalent doses. Both protein denaturation and HRBC membrane assays revealed maximal efficacy for stem extracts and diclofenac sodium at 100 μg/ml. These findings suggest that the phytoconstituents present in the ethanolic stem extract of A. echioides contribute to its anti-arthritic activity.

Acknowledgments

I extend my heartfelt thanks to the faculty and staff members of the Department of Botany for providing the necessary facilities and assistance. Finally, I acknowledge all the researchers whose work has contributed to the foundation of this study.

Conflict of interest

The authors declare that they have no conflict of interest or personal relationships that could have appeared to influence the work reported in this paper.