INTRODUCTION:

The word inflammation comes from the Latin word inflammare, means to burn. Inflammation is a response of the tissues to an injection, irritation (or) foreign substances. It is a part of host’s defence (or) inflammation, a defensive reaction to injury with classical signs of warmth, reddening, pain, swelling and loss of function, is of acute or chronic type Inflammation is normal and necessary protective response to the harmful stimuli such as infectious agents, antigen-antibody reactions, thermal, chemical, physical agents, and Ischemia. It is caused by a variety of stimuli, including physical damage, UV irradiation, microbial attack, and immune reactions. The classical key features of inflammation are redness, warmth, swelling, and pain. Inflammation cascades can lead to the development of diseases such as chronic asthma, arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis. Many of these diseases are debilitating and are becoming increasingly common in our ageing society^1-5.

Medicinal plants can be important source of previously unknown chemical substances with potenttherapeutic effects. Herbal medicines are in great demand in the developed as well as developing countries for primary health care because of their wide biological and medicinal activities, higher safety margins and lesser costs. Natural products in general and medicinal plants in particular are believed to be an important source of new chemical substances with potential therapeutic effects^6-8. Plant extracts as well as their primary and secondary metabolites have important therapeutic role in the treatment of many human diseases. Medicinal plants have a traditional medicine or folk medicine practise based on the use of plants and plant extracts. Many herbs synthesize substances that are useful for the maintenance of human health. Phytochemicals not only have protective or disease preventive properties but also protect humans from a host of diseases. Major constituents of more than 50% of all the drugs in clinical use are natural products and their derivatives. There has been an growing interest in the study of medicinal plants as natural products in diverse part of the world. Medicinal plants containing active chemical constituents with higher antioxidant property play an important role in the prevention of various degenerative diseases^9-10.

Plants have been always used as medicine by mankind to treat health-threatening diseases and still popular to obtain new drug candidates as it is the oldest medical practice for humans^11-15. The use of botanical natural health products are on the increase all over the world. It is known that almost 80% of the populations in developing countries rely on the traditional medicine, mainly composing herbal prescriptions. Abrus precatorius is a woody twinning plant with characteristic red seeds with black mark at the base. It is native to India. It is a beautiful, much branched, slender, perennial, deciduous, woody, twinning or climbing herb^16-19.

Traditional uses of Abrus Precatorius:

Leaves:

The leaves of the herb are used to cure fever, cough and cold, the paste of leaves and seeds are applied for the greying of hair^20-22.

Roots:

Paste of roots is used to cure abdominal pains, tumours and also for abortion, root is chewed as a snake bite remedy, the roots are used to treat jaundice and haemoglobinuric bile, Hot water extract of fresh root is an anti-malarial and anti-convulsant, Decoction of dried root is used to treat bronchitis and hepatitis.

Seeds:

Dry seeds of A. precatorius are used to cure worm infection, in veterinary medicine, it is used in the treatment of fractures, Seeds have also the potential of good insecticide and antimicrobial activity^23-25.

Biological and Pharmacological Action:

During past several years Abrus Precatorius is gaining lot of interest according to researchers’ point of view. Recently many pharmacological studies have been conducted on Abrus Precatorius^26-28. A summary of the findings of these studies performed is presented below: Anti-microbial activity, Hepatoprotective, Immunomodulator, Anti- Diabetic, Anti-Oxidant, Anti-Ulcer, Anti-alleric, Anti-tumor, Anti-bacterial, Anti-fungal, Gastro protective activity, Anti-inflammatory^29-31

MATERIALS AND METHODS:

Collection of sample:

Fresh Abrus precatorius seeds were collected from Kistapuram, khammam district, Telangana during June 2018. They were washed with distilled water twice. Then they were dried and powdered.

Drugs and chemicals:

Folinciocalteus phenol reagent, bromocresol green solution, egg albumin, diclofenac, HRBC solution are the samples obtained from Sree Srinivasa Scientifics.

PREPARATION OF EXTRACTS:

250g of dried seed powder were suspended in 1liter of hydro alcohol (methanol: water 70:30). Extraction was done at 80⁰C by using soxhlate apparatus for 3 hour 30 minutes. Followed by filtering of the extracts using what man filter paper no 1. Extract was then concentrated at 80⁰C for 3hours to form a semisolid form and they were transferred into sterile wide mouthed bottles and refrigerated until used.

Physicochemical parameters:

Phytochemical analysis was carried out for saponins, flavonoids, cardiac glycosides, terpenoids, steroids, tannins, phenol, anthraquinone. Alkaloids, and tannins was performed as described by authors. wagners and hagers reagents were used for alkaloid foam test for saponins. Killer-killani test for cardiac glycosides, salkowski test for terpenoids, sulphuric acid for steroids, chloride and gelatin for tannins, ferric chloride for phenol, hexane and diluted ammonia for anthraquinone test, all these experiments were carried out for hydro alcoholic seed extract of abrus precatorius.

Evaluation of Anti- inflammatory activity:

Membrane stabilization method:

Preparation of red blood cells (RBCs) suspension: The blood was collected from healthy human volunteer who has not taken any NSAIDs (Non- steroidal anti-inflammatory drugs) for two weeks prior to the experiment and transferred to the centrifuge tubes. The tubes were centrifuged at 3000rpm for 10minutes and were washed 3 times with equal volume of normal saline. The volume of blood was measured and re constituted as 10% v/v suspension with normal saline.

Hypotonicity induced haemolysis:

Different concentrations of extract (100-800µg/ml), reference sample and control were separately mixed with 1ml of phosphate buffer, 2ml of hyposaline and 0.5ml of HRBC suspension. Diclofenac sodium was used as a standard drug. All the assay mixtures were incubated at 37ºC for 30 minutes and centrifuged at 3000rpm. The supernatant liquid was decanted and haemoglobin content was estimated by a spectrophotometer at 560nm. The experiment was performed at triplicate. The percentage haemolysis was estimated by assuming the haemolysis produced in the control as 100%.

% Protection = 100- (OD of sample/OD of control) ×100

Inhibition of albumin denaturation method:

The anti-inflammatory activity of Abrus precatorius was studied by using the method of inhibition of albumin denaturation technique. The reaction mixture was consists of 1ml of tests extracts (100µg- 800µg) and 1ml of 1% aqueous solution of bovine albumin fraction. The samples were incubated at 37±1°C for 15 min and then denaturation was induced by boiling at 60°C for 10 min, after cooling the samples the turbidity was measured at 640nm. The experimentwas performed at triplicate. The percentage inhibition of albumin denaturation was studied by using following formula:

Percentage inhibition = (Absorbance of Control-Absorbance of Sample) × 100/Absorbance of control.

RESULTS AND DISCUSSION:

Physicochemical screening:

S. No

Physicochemical parameters

Seed powder

Extractive values:

Hexane

Chloroform

Ethyl acetate

Acetone

Methanol

Water

Methanol: Water

Swelling Index

Foaming Index

Ash values

Total ash

Water soluble ash

Acid insoluble ash

Loss on drying

1.59%

2.4%

4.55%

2.94%

9.02%

15.69%

12.5%

<100

3.04%

58%

2.14%

8.4%

Phytochemical Screening:

Table No: 2: Preliminary phytochemical analysis of hydro-alcoholic seed extracts of Abrus precatorius.

Tests	Hydro-alcoholic seed extract
Alkaloids	++
Triterpenoids	++
Tannins	+
Phenols	+
Flavonoids	+
Saponins	++
Carbohydrates	-
Proteins	+
Glycosides	+
Coumarins	+

(+++ = more intense, ++= intense, += present, - = absent)

Preliminary phytochemical screening of the hydro alcoholic seed extract of A. Precatorius reveals the presence of Alkaloids, Tannins, Glycosides, Saponins, Flavonoids and Terpenoids. Different doses of the extracts were screened for their activity mainly due to the presence of flavonoids respectively as shown in table 1 and 2.

QUANTITATIVE ESTIMATION:

Table: 3 Results for the quantitative estimation of the hydro-alcoholic seed extract of Abrus precatorius.

Test type	Seed
Total Flavonoid content	10.76±0.00(mg QE/g)
Total Alkaloid content	51.56±0.028(mg A/g)
Total Phenolic content	34.91±0.030(mg GA/g)

Table: 4 percentage membrane stabilization activity of the hydro-alcoholic seed extract of Abrus precatorius.

Concentration (µg/ml)	% Membrane stabilization
Concentration (µg/ml)	Hydro Alcoholic Seed Extract	Diclofenac sodium
100	10.71	8.2
200	41.07	33.2
400	50	38.4
800	64.2	50.3

Figure no 1: Anti inflammatory activity of Abrus precatorius seed Extract by using Membrane stabilization method

The HRBC membrane stabilization has been used as a method to study the invitro anti- inflammatory activity because the erythrocyte membrane is analogous to the lysosomal membrane and its stabilization implies that the extract may well stabilize lysosomal membranes. Stabilization of lysosomal membrane is important in limiting the inflammatory response by preventing the release of lysosomal constituents of activated neutrophil, such as bacterial enzymes and proteases, which causes further tissue inflammation and damage upon extra cellular release. The lysosomal enzymes released during inflammation produce a various disorders. The extra cellular activity of these enzymes are said to be related to acute or chronic inflammation. The non-steroidal drugs act either by inhibiting these lysosomal enzymes or by stabilizing the lysosomal membrane.

The hydro-alcoholic root, seed and leaf extracts of Abrus precatorius showed dose dependant membrane stabilization activity. The hydro-alcoholic root extract of Abrus precatorius has showed better membrane stabilization activity i.e., 64.2% at the concentration 800µg/ml as compared to the membrane stabilization activity of standard drug (Diclofenac sodium) i.e., 8.2% at the concentration of 100µg/ml as shown in figure no 1 and table no 4.

Anti inflammatory activity of Abrus precatorius seed Extract by Percentage inhibition of albumin denaturation method

Table 5: Percentage inhibition of albumin denaturation of the hydro- alcoholic seed extract of Abrus precatorius.

Concentration (µg/ml)	% inhibition of albumin denaturation method
Concentration (µg/ml)	Hydro Alcoholic Seed Extract	Diclofenac sodium
100	9.09	8
200	18.1	13.8
400	36.3	24.3
800	45.4	30.3

Figure 2: Anti inflammatory activity of Abrus precatorius seed Extract by inhibition of albumin denaturation method

Protein denaturation is a process in which proteins lose their tertiary structure and secondary structure by application of external stress or compound, such as strong acid or base, a concentrated inorganic salt, an organic solvent or heat. Most biological proteins lose their biological function when denatured. Denaturation of proteins is a well-documented cause of inflammation. As part of the investigation on the mechanism of the anti-inflammation activity, ability of plant extract to inhibit protein denaturation was studied. It was effective in inhibiting heat induced albumin denaturation.

The hydro-alcoholic root, seed extract and leaf extracts of Abrus precatorius showed dose dependant inhibition of albumin denaturation activity. The hydro-alcoholic root extract of Abrus precatorius has showed better inhibition of albumin denaturation activity i.e., 45.4% at the concentration 800µg/ml as compared to the inhibition of albumin denaturation activity of standard drug (Diclofenac sodium) i.e., 8% at the concentration of 100µg/ml.

Hydro alcoholic extract has significant Anti-inflammatory activity when compared to standard Diclofenac sodium. Membrane stabilization and percentage inhibition of albumin denaturation methods of hydro alcoholic extract was 10.71,41.07,50,64.2 and 9.09,18.1,36.3,45.4 respectively at concentrations 100, 200, 400, 800 µg/ml for the seed extract. and these are 8.2,33.2,38.4,50.3 and 8,13.8,24.3,30.3 respectively at concentrations 100, 200, 400, 800 µg/ml for Diclofenac sodium as shown in table no 5 and figure no 2.

CONCLUSION:

The work states that the presence of Flavonoids, Terpenoids and in the extract of Abrus Precatorius Linn was responsible for its Anti-inflammatory activity. Hydro alcoholic extract was shown significant Invitro activities like Membrane stabilization and percentage of inhibition of albumin denaturation methods. It is interesting to observe the results of Anti-inflammatory effect of hydro alcohol extract. But further investigations on the isolation of active compounds present in the extracts and in vivo studies are necessary to identify a potential chemical entity for clinical use. Hydro-alcoholic root extract of Abrus precatorius showed maximum membrane stabilization and inhibition of albumin denaturation activity in comparison to hydro alcoholic seed and leaf extract of Abrus precatorius. The results of the membrane stabilization activity and the inhibition of albumin denaturation activity indicates that the hydro-alcoholic root, seed and leaf extract of Abrus precatorius shows more membrane stabilization when compared to inhibition of albumin denaturation.

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Received on 28.11.2018 Modified on 31.12.2018

Asian J. Res. Pharm. Sci. 2019; 9(1):50-54.

DOI: 10.5958/2231-5659.2019.00008.0