In vitro Anti-inflammatory Properties of Various Extracts of Andrographis echioides

Background: Andrographis echioides (Acanthaceae) which is commonly known as false water willow is a herb commonly found throughout India. Some of them are medicinally important. The plants from genus Andrographis are used in goiter, liver diseases, fever, fertility problems, bacterial, malarial, helminthic fungal, diarrhea and larvicidal disorders. Materials and Methods: The in-vitro anti inflammatory properties of aqueous, ethanolic, chloroform extracts were evaluated by Protein denaturation assay. Aspirin was a positive control. The experiment was done in triplicates and percent inhibition of protein denaturation was calculated. The data were analysed statistically using one way analysis of variance ( ONE-WAY ANOVA). Duncan's multiple tests were used to analyse the statistical significance between groups. The level of significance was considered at the levels of p<0.005. Results: There was a dose-dependent increase in protease inhibition of protein denaturation in Original Research Article Javith et al.; JPRI, 33(62A): 517-523, 2021; Article no.JPRI.74362 518 three extracts from the concentration ranging from 100 to 500 ug/ml. All the three extracts of Andrographis echioides showed better in-vitro anti-inflammatory effect compounds. Conclusion: The study concluded an innovative finding that aqueous, ethanolic, chloroform of Andrographis echioides possessed potent in vitro anti inflammatory effect attributed to its flavonoid, phenyl glycosides compounds.


INTRODUCTION
Plants are important sources of medicines and presently about 25% of pharmaceutical prescriptions within the US contain a minimum of one plant-derived ingredient. In the last five decades, roughly 121 pharmaceutical products were formulated to support the traditional knowledge obtained from various sources [1). Phytomedicine or phytotherapy or botanical medicine was collectively called herbal medicine. It refers to herbal materials or medicinal preparations that contain plant parts as active ingredients [2]. The lack of scientific evidence in herbal medicines, when compared to that of modern medicine, gradually made herbal medicine become unpopular among people [3]. Since ancient times people were dependent upon plants for their food, shelter and medicine.  [11].
Andrographis echioides (Acanthaceae) which is usually referred to as false water willow is a very rare herb commonly found throughout India [12]. Andrographis echioides L. is an annual herb present throughout South Indian. However, information on the chemical composition and bioactivity of this species is extremely rare [13]. The genus Andrographis is a native of India containing 28 species of small annual shrubs essentially distributed in tropical Asia. Some of them are medicinally important [14]. The plant from genus Andrographis is usually utilized in goiter, liver diseases [15], fertility problems, bacterial [16], malarial and fungal disorders [17] . Leaf juice boiled with coconut oil has controlled the falling and graying of hair [18] Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may be inactive to many tissues and organs and the synovial joints [19]. Statistically analyzed exhibit 1% of the population in the world affected by rheumatoid arthritis and females are affected three times more than the male. Allopathic treatment of Rheumatoid arthritis is prone to many complications. So, the present study planned to find a novel herbal antibiotic for the treatment of inflammatory conditions.

Chemicals
All chemicals and reagents used in this study were purchased from Sigma Chemical Company St. Louis, MO, USA; Invitrogen, USA; Eurofins Genomics India Pvt Ltd, Bangalore, India; New England Biolabs (NEB), USA; Promega, USA.

Inhibition of albumin denaturation
The anti-inflammatory activity of the plant extract was studied by the inhibition of albumin denaturation technique which was studied according to the methods of Mizushima and Kobayashi, 1968 and Sakat et al (2010) followed with minor modifications. The reaction mixture consisted of test extracts and 1% aqueous solution of bovine serum albumin, pH of the reaction mixture was adjusted using a small amount of 1N HCl to dissolve the mixture.The plant extract with increase in concentration (100 to 500 µg/ml) were incubated at 37 ºC for 20 min and later heated to 51 º C for 20 min, after the samples were cooled,the turbidity was measured at 660nm.( UVVisible Spectrophotometer Model 371, Elico India Ltd) The experiment was performed in triplicate. In this study, Aspirin was used as a standard anti-inflammatory drug.

Statistical Analysis
The data were analysed statistically using one way analysis of variance (ONE-WAY ANOVA). Duncan Multiple range test was used to analyze the statistical significance between groups. The levels of significance were considered at the levels of p<0.05.

RESULTS
The present study revealed that there was a dose dependent increase in inhibition of protein denaturation in aqueous, ethanolic and chloroform extract at a concentration ranging from 100 to 500 ug/ml. (p<0.05). The results showed that 400 ug/ml and 500 ug/ml showed maximum inhibition. (p<0.05) (Fig. 1-3). On the other hand, the chloroform extract possessed a very good amount of flavonoids and steroids [20]. Flavonoids have antioxidant activities and they have the property of preventing oxidative cell damage and carcinogenesis [21]. Steroids, abundant in many plants, have been shown to have hypercholesterolemia effects, diuretics and also exhibit anti-leukemic, antipyretic, and derivatives of steroids are active as anticancer and antiviral agents [22]. Steroids have been reported to stimulate menstrual discharge and diminish the secretion of milk [23]. Phytochemicals have long been recognized to possess many properties including antioxidant, anti-allergic, antiinflammatory, anti-viral, anti-proliferative and anti-carcinogenic [24,21]. The presence of free radicals and oxidative stress are implicated in many diseases like stroke, asthma, cancer, atherosclerosis, diabetes and arthritis. There had been an increasing interest in plant-derived antioxidants, because it could protect our body from free radical damage, diabetes and agerelated disorders [25,26].
Phytochemicals derived from plant products function as a prototype to develop less toxic and simpler medicines in controlling the expansion of microorganisms. These active compounds either might act alone or in combination to inhibit bacterial growth [27]. Many previous studies indicated that phenolic compounds stop many diseases from pathogenic invasion [20]. Nowadays efforts are focused on plants because of their usage of historical times and the world's population rely on plants for the treatment of infections and noninfectious diseases [28]. However, isolation of specific phytoconstituents and screening for biological activities will definitely give better results [29]. From this present study, it might be concluded that A. echioides contains various bioactive compounds hence, it can be considered as a plant of phytopharmaceutical importance. Therefore, these plants seem to serve as prospective material for advance development of plant-based anti-inflammatory agents.
Phytochemical investigation of the entire plant of Andrographis echioides leads to the presence of two new oxygenated flavonoids, phenyl glycosides alongside 30 non-structures [30,31]. These bioactive compounds offer the plant a good anti-inflammatory activity. The extract of the plant Andrographis echioides was rarely available and costly. Hence it was hard to conduct and complete the research.

CONCLUSION
In conclusion, different extracts of A. echioides can be very effective anti-inflammatory and it could protect biological systems against the oxidative stress including aging, cancer, diabetes and cardiovascular disorders. Further investigations that can identify and characterise the specific active components of the plant extract can offer better explanations for its antiinflammatory potential.

NOTE
The study highlights the efficacy of "herbal medicine" which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.

DISCLAIMER
Authors have declared that no competing interests exist. The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.