In vitro antipsoriatic study of successive solvent extracts of Barleria gibsoni Dalz. using HaCa T keratinocyte cells

Firoj A. Tamboli*, Harinath N. More

Department of Pharmacognosy, Bharati Vidyapeeth College of Pharmacy, Near Chitranagari, Kolhapur -416013

*Corresponding Author E-mail: firojtamboli143@gmail.com, firoj.tamboli@bharatividyapeeth.edu

ABSTRACT:

The present study was to investigate the in vitro antipsoriatic activity of the successive solvent extracts of Barleria gibsoni Dalz. leaves against HaCa T keratinocyte cells. Asiaticoside was given as reference drug. The plant material was dried under shade, powdered and extracted with different solvent extract like Petroleum ether, chloroform, ethyl acetate and ethanol. Preliminary phytochemical screening was done. 50% cell growth inhibition (IC50) of successive solvent extract of Barleria gibsoni and Asiaticoside was 576.82 µg/ml and 460.13µg/ml against the HaCa T keratinocyte cells. Petroleum ether and ethanol extract of Barleria gibsoni and Asiaticoside was effective cytotoxic drugs against HaCa T keratinocyte cells. It is due to the extraction of active compounds in the Petroleum ether and ethanolic extract. Therefore, it is suggested that evaluation of active constituents and clinical evaluation of petroleum ether extract of Barleria gibsoni would give a encouraging lead in the successful management of psoriasis.

KEYWORDS: Barleria gibsoni Dalz. Acanthaceae, HaCa T keratinocyte cells, Antipsoriatic activity, Asiaticoside.

INTRODUCTION:

Psoriasis is a chronic life-long inflammatory disease that primarily affects the skin, musculoskeletal system, the gastrointestinal system and the eye.¹Being an autoimmune disorder no diagnostic tests available for identification of psoriasis.² However, the current treatments have not fully met the needs of the sufferers, largely due to the side effects so often associated with various therapies. Also, a large proportion of patients would develop drug resistance after long term drug exposure.³ Natural remedies seem promising in the management of wide range of dermatological conditions including psoriasis vulgaris.⁴ Barleria gibsoni Dalz. (Family Acanthaceae) is widely distributed throughout Africa, India, Sri Lanka and tropical Asia. It is commonly known as Neel koranti. Juice of the leaves is used in ulcer, cataract and fever. The dried bark is used in cough treatment and the leaves chewed to relieve toothache. The paste of the root is applied to disperse boils and glandular swellings ⁵.

It exhibits several medicinal properties. Leaves are also used by some tribal communities for the treatment of piles and to control irritation. Plant is also used in stiffness of limbs, enlargement of scrotum and sciatica^6-9. Therefore, to investigate its traditional use in the treatment of psoriasis, so that extracts investigate in vitro antipsoriatic study of successive solvent extract of Barleria gibsoni using HaCa T keratinocyte cells.

MATERIALS AND METHODS:

Collection and Identification of plant material

The fresh leaves of the Barleria gibsoni were collected from the local region of Satara (Maharashtra) and authenticated by taxonomist at Botanical survey of India, Pune, Maharashtra. A voucher specimen has been deposited at the herbarium of Botanical survey of India, Pune. (BSI/WRC/Tech/2013/FAT 01 dated 27^thDecember, 2013)

Preparation of Extracts

The powdered plant material was used for extraction by Soxhlet apparatus at elevated temperature (65^oC) using Petroleum ether, Chloroform, Ethyl acetate and Ethanol consecutively with 500 ml of each solvent. After each extraction the plant material was dried and used again for the next extraction. Extraction was considered to be complete when the plant materials become exhausted of their constituents that were confirmed from cycles of colorless liquid siphoning in the Soxhlet apparatus.

Maintenance of cell lines

Human HaCaT keratinocytes were obtained from National Centre for Cell Science (NCCS), Pune, India. Stock cells of these cell lines were cultured in Dulbecco’s Modified Eagles Media (DMEM), supplemented with 10% FBS (Fetal bovine serum). Along with media cells were also supplemented with 5 % HBSS, penicillin, streptomycin and Amphotericin – B, in a humidified atmosphere of 5 % CO₂ at 37 °C until confluence reached. The cells were dissociated with 0.2 % trypsin, 0.02 % EDTA in phosphate buffer saline solution. The stock cultures were grown initially in 25 cm² tissue culture flasks, then in 75 cm² and finally in 150 cm² tissue culture flask and cytotoxicity experiment were carried out in 96 microtitre well- plates.

The cells were seeded at a concentration of 1.0×10⁵cells/mL in a 96 well microtitre plate and grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (BioWest).

Phytochemical test

Preliminary phytochemical analysis of the extracts was performed by standard chemical tests.^10,11

Cytotoxicity assay

In vitro anti-psoriatic activity was carried out in HaCaT human keratinocyte cell line¹². After 24 h, the supernatant was decanted and the monolayer was washed once. Then 100 µL of test substance in various concentrations was added to the cells in microtitre plates. Test compounds were prepared in dimethyl sulphoxide (DMSO) and then diluted with DMEM; the final concentration of DMSO was 0.2% in the culture medium. Each sample concentration was tested in triplicates. Controls were performed with DMSO or medium alone. Asiaticoside (Sigma) was used as positive control. The plates were then incubated at 37°C for 3 days in 5% CO₂ atmosphere. Antiproliferant activity was assessed by performing the Sulphorhodamine B (SRB) assay. Cells were fixed by adding 25 µL of ice-cold 50% trichloroacetic acid on top of the growth medium and the plates were incubated at 4°C for 1 h, after which plates were washed to remove traces of medium, drug and serum. SRB stain (50 µL; 0.4% in 1% acetic acid) (Sigma) was added to each well and left in contact with the cells for 30 min after which they were washed with 1% acetic acid, rinsing 4 times until only dye adhering to the cells was left. The plates were then dried and 100 µL of 10 mM Tris buffer (Sigma) was added to each well to solubilise the dye. The plates were shaken gently for 5 min and absorbance was measured at 550 nm using a micro plate reader (BioTek, USA).

% viability = (AT-AB)/ (AC-AB) x 100

Where, A_{T =} Absorbance of treated cells (drug); A_{B =} Absorbance of blank (only media) and A_C
= Absorbance of control (untreated)

% cell growth inhibition / % cytotoxicity = 100 – % cell survival

Statistical analysis

All the values of % cell growth inhibition was replicates of three independent observations (n=3). The 50% cell growth inhibition (IC₅₀) was determined by interpolating concentration (X-axis) Vs % cell inhibition (Y-axis) by linear regression equation using Microsoft Excel, 2007, Microsoft Corporation, USA application.

RESULTS AND DISCUSSION:

Preliminary Phytochemical Screening:

In the present study, preliminary phytochemical screening study of extracts under consideration was done to identify the presence of various bioactive components. Preliminary phytochemical screening of the crude extracts of leaves of Barleria gibsoni showed the presences of different phytochemical constituents are as summarized in the Table 1.

Table. 1: Result of phytochemical screening of Successive solvent extracts of leaves of Barleria gibsoni

Phytochemical Test		Successive solvent extracts of leaves of Barleria gibsoni
Phytochemical Test		Petroleum ether extract	Chloroform extract	Ethyl acetate extract	ethanol extract
Alkaloid	Mayer’s test	+	+	-	+
	Hager’s test	++	+	+	+
	Wagner’s test	++	+	+	+
	Dragandroff’s test	++	+	+	+
Carbohydrate		-	_	+	_
Flavonoid		-	+	+	+
Glycoside		+	+	+	+
Saponin		-	_	+	+
Steroid		+	+	+	-
Tannin		_	_	_	_

“+” indicates presence, “++” indicates strong presence, “-“indicates absence

Table. 2: % cell inhibition of Successive solvent extracts of leaves of Barleria gibsoni on HaCaT human keratinocyte cell line

Conc. µg/ml	HaCaT human keratinocyte cell line
Conc. µg/ml	Petroleum ether	Chloroform	Ethyl acetate	Ethanol	Standard
10	46.42	21.30	29.52	38.23	78.57
25	55.42	35.40	31.56	41.20	80.11
50	65.47	38.10	36.66	45.23	83.33
75	71.26	41.20	42.13	51.12	85.47
100	73.80	45.20	48.23	55.35	90.89
IC₅₀	55.85	88.80	86.50	72.35	41.98

Fig. 5: % cell inhibition of standard drug on HaCaT human keratinocyte cell line

Due to disturbances in leukotriene homoeostasis can result in inflammatory responses as diverse as psoriasis, rheumatoid arthritis and inflammatory bowel disease. The presence of cysteinyl leukotrienes (slow-reacting substances) is reported in inflammatory diseases such as psoriasis.¹³ It is well known underlying mechanisms of psoriasis. Since steroid, quercetin inhibit leukotriene synthesis and histamine release, as well as act as superoxide scavengers, they could have a palliative effect on inflammation.^14,15 Since flavonoids and steroids are well effective for psoriasis and our preliminary phytochemical investigation revealed the presence of flavanoids in ethanolic extract of Barleria gibsoni. Psoriasis is a disease resulted from the hyper proliferation and abnormal differentiation of keratinocytes.¹⁶ A successful antipsoriatic drug that targets the epidermis is defined as a compound that ideally shows low toxicity and restores skin homeostasis by suppressing keratinocyte hyper proliferation, abnormal differentiation, or both.¹⁷ The present study aimed to investigate the anti-proliferative properties of different extracts of Barleria gibsoni, which were selected from the traditional information for their anti-proliferative effects against keratinocytes using cultured HaCaT cells as a psoriasis-relevant experimental model.

CONCLUSION:

From the present study it can be said that Petroleum ether and ethanol extract of Baleria gibsoni has in vitro antipsoriatic activity and it will be beneficial in the management of psoriasis.

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Received on 24.10.2015 Modified on 06.11.2015

Research J. Pharm. and Tech. 8(11): Nov., 2015; Page 1566-1569

DOI: 10.5958/0974-360X.2015.00279.6