Liquid – Liquid Fractionation and Evaluation of In-Vitro Anti-Inflammatory Activity of Ethanol Extract of Leucas Aspera

Inflammation is a biological defense mechanism that enables living cells to protect themselves against diseases such as bacteria, fungi, viruses, physical agents, and defective immune function, inflammation may be acute (initial inflammation or chronic (out of proportion of protection damage) types. Factors such as redness, swelling, pain, loss of function of cells, and heat are common symptoms of   inflammation. Human living cells naturally develop protective mechanisms in response to body inflammation due to microbial infections, mechanical injuries, and burns stimuli Both acute and chronic inflammatory responses play a significant role in the natural defense mechanisms of the human body’s inborn immune system to maintain human health. Its main aim is to stimulate living cells to eliminate harmful agents and remove damaged tissues to heal the affected parts. The inflammatory response is effective by secretion of different mediators responsible for the initiation, progression, persistence, regulation, and resolution of inflammation effects.¹ Inflammation can be classified as either acute or chronic. Acute inflammationis the initial response of the body to harmful stimuli and is achieved by the progressive movement of plasma and leukocyte-like constituents from the blood  into the injured tissues/locations. Chronic inflammation leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous breakdown and healing of the tissue from the inflammatory process.²in our body inflammation is regulated by numerous signalling  pathways forming a complex system, in general, this complex biological response leads to the restoration of  homeostasis, for the treatment of inflammation, people have been using non-steroidal anti-inflammatory drugs (NSAIDs) as medicines. However the increasing side effects such as heart attack and strokes due to these drugs are  main reasons  needed to replace synthetic drugs with minimal risk-causing plant-based medicines.³ Medicinal plants are the only source for the treatment of diseases in ancient days and since then numerous herbs and plants have been recognized as a medicinal plants because of their potency to cure ailments. The newly discovered and the existing medicinal plants are being screened for many diseases and to identify significant therapeutic importance.⁴ Leucas aspera is a perennial herb found commonly in tropical regions, it is an herbaceous aromatic weed belonging to the family Lamiaceae (Labiatae), traditionally it is known as “Thumbai” in Tamil and “Dronapushpi” in Sanskrit and It is used as medicinal plant since ancient times.The whole plant is traditionally important because it has many therapeutic values, Leucasaspera flowers are used as stimulant, expectorant (treat coughs), asperients (relieve constipation), and diaphoretic (inducing perspiration), insecticide and the flowers are mixed with honey and given to children to treat cold. The leaves are used as insecticides and mosquito repellent, the leaf juices are considered as a remedy for chronic rheumatism, psoriasis and other chronic skin eruptions, for stomach pain and indigestion the plant extract was given with honey.⁵ The extensive literature review on Leucas aspera revels that the plant as many phytoconstituents like Polyphenols, Flavonoids, Terpines, Alkaloids and essential oils etc , so the present project work has been taken to screen the anti-inflammatory properties of  various fractions of   70%  ethanol extract  of plant by In-vitro protein denutaration  (egg albumin)  method.

Review of Literature

Plant profile:

L. aspera is a small herbaceous erect plant with a free blooming nature and flowering in the months of August to September. It is pungently aromatic herb and grows abundantly in the high land crop fields, roadsides, homesteads and fallow lands all over India the plant genus Leucas belongs to Lamiaceae family and embraces about 80 species, which are widely employed to cure many diseases by the traditional healers worldwide.

Vernacular Names

Sanskrit: Dronapushpi, Chitrapathrika, Chitrakshup

Kannada: Tumbi

Punjabi: Guldor         

Bengali: Darunaphula, Hulkasha

Gujarati: Kulnnphul

Hindi: Gomamadhupati

Sindhi: Kubo

Maharashtra: Bahuphul

Telugu: Thummichittu

Leucas aspera is an aromatic herb distributed in India, Philippines, The plains of Mauritius and Java, Tropical Asia and Africa, It grows as a competitive weed and as annual herb found in highland crop fields, fallow lands, cultivated fields, sandy soils, wastelands and roadsides, grows upto 15 – 60 cm height with quadrangular stem and branches.⁶

Botanical description: Leucas aspera is an annual, branched, herb erecting to a height of 15-60 cm with stout and hispid acutely quadrangular stem and branches. Leaves are sub-sessile or shortly petiolate, linear or linearly lanceolate, obtuse, pubescent up to 8.0 cm long and 1.25 cm broad, with entire or crenate margin; petiole 2.5-6 mm long; flowers white, sessile small, in dense terminal or axillary whorls; bracts 6 mm long, linear, acute, bristle-tipped, ciliate with long slender hairs; calyx variable, tubular, 8-13 mm long; tube curved, contracted above the nutlets, the lower half usually glabrous and membranous, the upper half ribbed and hispid; mouth small, very oblique, not villous, the upper part produced forward; teeth small, triangular, bristle-tipped, ciliate, the upper tooth being the largest. Corolla 1 cm long; tube 5 mm long and pubescent above, annulate in the middle; upper lip 3 mm long, densely white-woolly; lower lip about twice as long, the middle lobe obviate, rounded, the lateral lobes small, subacute. Fruit nutlets, 2.5 mm long, oblong, brown, smooth, inner face angular and outer face rounded.^7,8.

METHODOLGY

Selection and Extraction of Plants Material-

The aerial part of Leucasasperawere generally collected around the fields of    Harapanahalli, Vijayanagara (D) Karnataka, INDIA. Authentication of Raw Plant Material is the basic Starting Priority in Developing of Botanical/Herbal Product. Lucas aspera Plant Material was authenticated by Prof. K Prabhu Sir, Dept. Of Pharmacognacy, S.C.S. College of Pharmacy, Harapanahalli, Herbarium is deposited in the Department of Pharmacognocy. Whole plant of Leucas aspera were dried under shade, mixed together & then made in to coarse powder with mechanical grinder and extracted by continuous hot extraction method in Soxhlet apparatus. The percentage yield was calculated for the extracts with reference to the crude material taken using the formula given below.

       
           
       
Fractionation of crude extract was carried out by suspending 73.54gm of extract in 200ml of water and then partitioning with hexane, chloroform and acetate in order of increasing polarity by using separating funnel. All the fractions including aqueous fraction were condensed using water bath. Fractions so obtained were hexane (13.61gm), chloroform (32.3 gm), ethyl acetate (1.23gm), ethanol (13.07gm) and aqueous (3.54gm).

Preliminary phytochemical screening 

The obtained fractions of the plant extract were subjected to preliminary phytochemical screening following the standard procedures described in the practical Pharmacognosy by C.K. Kokate⁹ and R.K. Khandelwal¹⁰ results are summarized in table no.

Inhibition of Protein Denaturation.

The anti-inflammatory activity of various fractions of 70% alcoholic extract of Leucas aspera were carried out according to the modified methods of  Mduranga¹¹ and  Sakatet al.¹² The anti-inflammatory activity of  various fractions of 70% alcoholic  extract can be determined by In- vitro  inhibition of the denaturation of egg albumin (protein).  0.2 mL of 1-2% egg albumin solution (from fresh hen’s egg/ or commercially available egg albumin powder), 2 mL of sample extract or standard (Diclofenac) at varying concentrations, and 2.8 mL of phosphate bufferer saline (pH 7.4) were mixed to form a reaction mixture of a total volume of 5 mL.

•  A total volume of 5 mL of the control was created by combining 2 mL of triple-distilled water, 0.2 mL of 1-2% egg albumin solution, and 2.8 mL of phosphate-bufferer saline.

 • The reaction mixtures were then incubated at 37±2°C for 30 min and will be heated in a water bath at 70±2°C for 15 min.

 • After cooling, the absorbance was measured at 660 nm by a suitable UV/Vis spectrophotometer using triple distilled water as the blank [1].

• The following equation was used to determine the % inhibition of protein denaturation

Table no 01 Percentage and Colour of the Extract

II. Fractionation

Fractionation of 70% Ethanol extract was carried out with different solvents with increasing order of polarity and fractions so obtained were given in the table no 02

Table no 02: Fractions of extract

III. Qualitative analysis of phytochemicals

The obtained extracts were tested for various chemical constituent The according to standard procedure and results are shown in the following table no 03

IV.  Inhibition of Protein Denaturation

There are many In-Vitro methods to estimate anti-inflammatory actions of the drugs in our present studies inhibition of protein (albumin) denaturation was carried out with different fractions of plant extract and results are represented in table no  04 and fig no   02   From our studies we found that all fractions shows inhibition of albumin denaturation with increasing order of concentration in that ethanol fractions shows more significant protection  of  inflammation  or  prevention of protein denaturation with minimum inhibition  23.4±0.15%  to maximum inhibition of  65.14±0.72%.from 20% to 100% concentration respectively.

Table no 04: In vitro protein denaturation of Different fractions of 70% ethanolic extract

Values are expressed in mean ± SE of 3 replicate