Screening of Complement Stimulating Activity of Lindernia madayiparense Extracts

 

Umakrithika Selvaraj¹*, Prabal Kumar Manna², Kannan Kamarajan²

¹Department of Pharmaceutical Chemistry, Swamy Vivekanandha College of Pharmacy,

Nammakkal - 637205, Tamil Nadu, India

²Department of Pharmacy, Faculty of Engineering and Technology, Annamalai University,

Annamalainagar - 608002, Tamil Nadu, India

 

ABSTRACT:

This study evaluates the influence of different extracts of newly identified Lindernia madayiparense from Linderniaceae on complement system. The similar species under this family are used in China and India to prevent and cure diverse ailments and to rejuvenate the immune system without any marketing standards. Various extracts of plant were evaluated through complement mediated hemolytic assay. Two plant extracts shows complement modulating property against alternative pathway of complement system, i.e. ethanol extract and decocted extract. These fractions showed significant complement stimulating activity compared to positive control used.

 

 

 

INTRODUCTION:

Complement is a collective term used to designate a group of plasma and cell membrane proteins that play key role in the host defense process¹. This complex system has three major functions:  to cause lysis to cells of microbes, to mediate the process of opsonization in phagocytosis, regulate the inflammatory and immune responses.  The physiological processes of complement activated through a sequential cascade like activation of enzymes normally present in their inactive forms in plasma. Two major pathways of complement activation operate in plasma are classic and alternative pathways. Classical pathway is initiated by antigen-antibody complexes and alternative pathway does not have an absolute requirement of antibody for activation.

 

Both pathways function through the interaction of proteins termed components and form a membrane attack complex, MAC through C3, which ultimately causes cell lysis. Besides directly damaging the invading organisms, the activation of complement may contribute to or even evoke pathological reactions in a variety of inflammatory diseases or degenerative diseases. Thus searching for the medicinal plants which can active upon the complement system gathered a extreme interest in the researchers.

 

Linderniaceae is a family of flowering plants and distributed throughout the world. Various species in this family acquire special place in several traditional medications used to prevent, cure different ailments and rejunvate immune system available in China and India². The plant, Lindernia madayiparense comes under this family was recently identified by MSSRF, Coimbatore. It is widely distributed in the laterite hills of Northern Kerala especially in the Madayipara, Kannur District, Kerala³. However there were no scientific evidences with reference to its biological activities. The earlier reports on the Lindernia species revealed the neuritogenic⁴, hepatoprotective⁵, antimicrobial and anti-inflammatory⁶, anti-tumor^7,8,9, antioxidant¹⁰, anthelminitic¹¹ and autophagy modulator¹² activities. These findings provide strong evidence and initiated the present research work with the hypothesis that Lindernia madayiparense also might exhibit diverse pharmacological activities. Hence, this research work was to evaluate immunomodulating activity of various, L. madayiparense extracts by suitable in-vitro method for the first time.  

 

MATERIALS AND METHODS:

Plant materials:

Wild crafted plant, Linderina madayiparanse was collected during its flowering season in the month of October to December, 2013 in Kannur District, Kerala, India. The plant material was identified and authenticated by botanist Mr. P. Biju, Assistant Professor, Government College, Kasaragod, Kerala, India.

 

Preparation of plant extracts:

Various extracts of L. madayiparense were prepared by two different extraction procedures. The methods were decoction and continuous hot extraction method using water, petroleum ether, ethylacetate and ethanol.

 

Preparation of 1% Rabbit erythrocytes (RbE):

Rabbit blood was collected freshly in a sterile bottle along with anticoagulant and centrifuged at 2000 rpm for 10 minutes. Supernatant was removed by decantation, cells were washed twice with Phosphate Buffer saline and twice with Veronal-buffered saline (VBS) and triethanol amine buffer (TEA) separately. 1% v/v suspension of RbE in VBS and TEA buffer was prepared separately by suspending washed erythrocytes in particular concentration^13,14.

 

In-vitro alternative complement pathway haemolytic activity:

The assay was performed in flat-bottom 96-well microtitre plate. Various dilutions of extracts and standard were prepared and transfer 100μl of each to micro-centrifuge tubes. Subsequently, 25μl of HS was added to each tube. All the tubes were incubated for 30 min at 37°C. After incubation, 25μl of RbE suspension was added to each tube and the tubes were again incubated at 37°C for 60 min. Then, the tubes were centrifuged at 1000 rpm for 6 min and the supernatant liquid was collected. From this, 50μl of the supernatant was transferred to flat-bottom microtiter plate and 200μl of distilled water was added and mixed well. The absorbance was measured at 412 nm in an ELISA automatic plate reader for all test dilutions and controls. The absorbance of complement blank was subtracted from absorbance values of test serum to get the corrected absorbance of test dilution^14,15,16,17.

 

Percentage haemolysis for each dilution was calculated using the following formula.

 

    (Corrected absorbance of test serum – Absorbance of blank)

% Haemolysis =------------------------------------------------X 100

         (Absorbance of Total lysis - Absorbance of blank blank)

 

 

ACH₅₀ was also calculated and tabulated.

 

RESULTS AND DISCUSSION:

Different dilutions of various extracts were prepared and analysed using VBS and TEA buffers. Haemolytic activity of ethanol extract and and decocted extract were slightly near that of standard, levamisole, an immunostimulant agent. Among the extracts, ethanolic extract and decotion exhibited highly significant concentration dependant AP haemolytic activity. The concentration required for 50% haemolysis (ACH₅₀) for levamisole, ethanol extract and decocted extract was found to be 353.28 ± 7.45 μg/ml, 878.35 ± 10.38 μg/ml and 971.56 ± 8.76 respectively with TEA buffer (Table 1) whereas ACH₅₀ was found to be  274.61 ± 3.12 μg/ml, 619.45 ± 8.21 μg/ml and 789.89 ± 5.32 respectively with VBS buffer (Table 2).

Table: 1. In-vitro Haemolytic activity of various extracts of the plant, L. madayiparense using TEA Buffer

S. No.		Percentage haemolysis
	Normal human serum	7.19±0.09
	Concentration (µg/ml)	Standard	Decocted extract	Pet ether extract	Ethyl acetate extract	Ethanol extract
1	1000	60.22±0.04	51.23±0.08	9.55±0.04	29.45±0.02	53.24±0.02
2	500	52.45±0.08	43.29±0.09	8.21±0.02	21.46±0.08	45.56±0.09
3	250	48.67±0.06	32.56±0.04	7.12±0.09	16.56±0.06	37.25±0.08
4	125	40.22±0.09	22.2±0.11	6.09±0.11	11.78±0.05	27.78±0.04
5	62.5	34.45±0.09	18.34±0.02	5.25±0.06	7.67±0.11	19.56±0.02
6	31.25	25.56±0.04	12.67±0.07	4.84±0.04	5.56±0.02	12.56±0.11
7	15.625	20.22±0.06	8.56±0.04	4.08±0.02	4.23±0.07	9.15±0.06
	ACH50	353.28±7.45	971.56±8.76	-	-	878.35±10.38

 

Table: 1.2. In-vitro haemolytic activity of various extracts of the plant, L. madayiparense using VBS buffer

S. No.		Percentage haemolysis
	Normal human serum	7.19±0.09
	Concentration (µg/ml)	Standard	Decocted extract	Pet ether extract	Ethyl acetate extract	Ethanol extract
1	1000	62.43±4.2	53.92±2.4	10.12±2.2	39.76±2.1	56.74±1.5
2	500	55.57±3.5	44.45±2.5	8.48±1.2	31.01±3.5	48.02±3.1
3	250	49.46±2.9	32.38±0.9	7.4±2.4	22.7±4.6	42.54±2.3
4	125	42.3±3.6	26.12±6.1	6.51±2.1	17.87±2.1	36.35±2.8
5	62.5	36.03±4.8	21.25±3.1	5.21±2.3	15.12±3.2	28.45±2.1
6	31.25	27.6±6.1	17.39±2.7	4.31±2.1	8.85±2.8	21.02±2.7
7	15.625	21.1±4.2	14.63±3.1	4.01±3.2	5.68±3.1	18.62±3.1
	ACH50	274.61±3.12	789.89±5.32	-	-	619.45±8.21

 

CONCLUSION:

The results suggested that the selected plant activates the Alternative pathway of complement system to some extent like other lindenia species which are used in immune enhancing herbal complex preparations. The present study revealed that the ethanolic extract and decoted extract of the plant, L. madayiparense in various concentrations exhibited the increased alternative complement pathway haemolytic activity to distinctive degree when compared with to the standard levamisole, in TEA and VBS. Furthermore the ACH₅₀ value found disclosed revealed that the ethanol extract has effectively increased the AP haemolytic activity than decocted extract.

 

Concerning illustration, the findings imply that the ethanol and decocted extracts of the plant, L. madayiparense might suppress specific adaptive immune system which leads in autoimmune disorder stimulated exorbitantly and stimulate innate immune system.

 

The outcomes from the findings recommend that the plant, L. madayiparense might be exhibited the immunostimulant action by invigorate innate immune system activity by alternative complement system and also it might be successful against cancerous cells. These findings support the cytotoxic effect of the plant, L. madayiparense.

 

ACKNOWLEDGEMENTS:

The authors are grateful to the Management and Department of Pharmacy, Annamalai University for their continuous encouragement and providing necessary facilities. Authors are also thankful to Mr. P. Biju, Assistant Professor, Government College, Kasaragod, Kerala for his support during the collection and identification of the selected plant.

 

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Accepted on 06.04.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4324-4326.