INTRODUCTION:

Cataract is a disease in which clouding develops in the capsule of the lens or in the crystalline lens of the eyes, leading to opacity of lens and obstructs the passage of light.^[1] It is a major complication of diabetes mellitus as increase in the level of glycosylated hemoglobin are significantly associated with the risk of cataract ; which is associated with aging.^[2] Though a wide variety of cataractogenic factors have been identified, yet the biochemical background of cataractogenesis still remains an unknown fact.

It is a multifactorial disease which occurs mainly due to formation of large protein aggregates in and around the lens. The prevalence of unoperated cataract increased with age and was higher in women than men.^[3]

Borreia hispida is one of the important plant of Rubiaceae family and is also a commonly used herb in Siddha medicine. Based on the literature survey of plant, it is used as an antidiabetic, antihypertensive, hepatoprotective, anti-inflammatory, antihyper-lipidaemic, analgesic, antifungal, anticancer and antioxidant^[4] Till now no research has been done on its anticataract activity; based on this fact this plant is selected for the present study and the probable mechanism is found out by which it shows its anticataract activity.

MATERIALS AND METHODOLGY:

Plant Materials:

The leaves of Borreria hispida, a perennial herb were collected from Thirumalpur in the Kancheepuram District, Tamilnadu and was authenticated by Dr. Jeyaraman, Botanist, Plant Research Centre, Tambaram.

Chemicals:

Sodium chloride, potassium chloride, Magnesium chloride, Calcium chloride, galactose and glucose. Human albumin, Folin cio calteau reagent were purchased from Southern Scientific Suppliers and the antibiotics Penicillin and streptomycin were purchased from Sudhagar Biochemicals Limited, Chennai

Preparation of extract:

The coarse leaves powder was extracted using 70% hydroalcoholic extract by hot percolation method using Soxhlet apparatus and the percentage yield was calculated.

Phytochemical Screening:

The phytochemical screening of hydroalcohholic leaves extract of Borreria hispida was done by standard method described by Harbourne ^[5]

Detection of Terpenoid:

By Maceration method, the species was extracted using chloroform, as solvent and the yield was dried for TLC of terpenoid.

TLC of triterpenoid:

In this TLC technique, toluene: ethyl acetate (93:7) mobile phase was used and the extract was diluted using ethyl ether and subjected to TLC standard procedure. The qualitative estimation of terpenoid was performed.

HPLC estimation of terpenoid:

The extraction of the test solution was done by dissolving in the solvent ratio (alcohol: water: hydrochloric acid in the ration of 50:20:8) with the mobile phase of, toluene: ethyl acetate (93:7 The test solution was injected to measure the areas for the major peak in the chromatograph and the amount of terpenoids present in the sample was determined.

Pharmacological study:

Invitro screening:

Lens Culture:

The goat lens were collected from slaughter house and immediately kept at 0-4⁰C in the laboratory. The lenses were removed by extracapsular extraction. .^[6]The damaged lens were discarded and the clear lens were incubated in artificial humour (NaCl 140mM, KCl 5mM, MgCl₂ 2mM, NaHCO₃ 0.5mM, NaH(PO₄)₂ 05mM,CaCl₂ 0.4mM, glucose 5.5mM) at room temperature for 72 hours at a pH of 7.8. Penicillin 32mg% and streptomycin 250mg% were added to culture media to prevent contamination. ^[7]Galactose in a concentration of 55mM was used to induce cataract. The dose of galactose was determined from the previous study on galactose induced cataract model.^[8]After incubation the lenses were homogenized to study protein content

GROUPING OF LENS:

A total of 16 lenses were divided into following categories containing 4 lens in each group.

GROUP 1: Normal lens

GROUP 2: positive control lens (55mM galactose)

GROUP 3: Test lens (1mg/ml Borreria hispida) + (55mM galactose)

GROUP 4: Standard lens (1mg/ml atropine) + (55mM galactose)

Estimation of total protein content:

The total protein content in the lens homogenate was determined by the method of Lowry et al ^[9] using bovine serum albumin (1mg/ml) as the standard and the absorbance was measured at 660nm.

Photographic evaluation:

Lenses were placed on a graph paper in such a way that the posterior surface touches the mesh and the pattern of mesh (number of squares clearly visible through lens) was observed through the lens as a measure of opacity of lens.^[10]

Estimation of total protein:

The total protein concentration was determined by Lowry’s photometric method, with human albumin (1 g/l) used for calibration.^[^11]

RESULT:

FIG 1: TLC of terpenoid in the hydroalcoholic leaf extract of Borreria hispida

Table:1 HPLC data of the terpenoids present in hydroalxcoholic extract of Borreria hispida leaves.

	Reten [min]	Area [mv. s]	Hight [mv]	Area [%]	Height [%]	W05 [min]
1	2.790	2400.835	53.110	38.3	83.6	0.49
2	9.190	2019.343	6.181	32.2	9.7	5.56
3	25.033	1847.582	4.207	29.5	6.6	7.64
	Total	6267.760	63.498	100.0	100.0

FIG 2: HPLC of terpenoids

FIG 3: Photocopy of lens

Fig:4 Estimation of Total protein in hydroalxcoholic extract of Borreria hispida leaves.

DISCUSSION:

The Advanced glycation end products and oxidative stress are the common cause to induce diabetic associated cataract which leads to blindness. Hyperglycemia, oxidative stress and UV radiation are the factors promoting cataractogenesis. ^[12]Galactose induced model is frequently used to study diabetic associated cataract. In this study, based on folkclaire data, Borreria hispida species was selected for anticataract activity.

The percentage yield of hydroalcoholic extract was found to be 1.25% w/w. The phytochemical screening revealed the presence of alkaloids, glycosides, flavonoids, saponins, terpenoids, phenols.

Borreria hispida belonging to Rubiaceae family has effective role against diabetes, In India the decoction of the leaves consumed for diabetes, headache and the internal injuries of nerves and kidney.^[13,14,15]

Among the phytoconstituents, tetracyclic triterpenoid has challenging role in inhibiting the protein aggregation and thereby preventing cataract formation in other plant species. Literature supports the presence of tetracyclic triterpenoid in Boreria hispida species. Based on this data, qualitative analysis of terpernoid was performed by TLC analysis followed by the quantification of terpenoids by HPLC techniques. The TLC of terpenoid was shown in fig;1, the pink bands represents the terpenoid content in the Borreria hispida species and the HPLC result shown the presence of three peaks which confirmed the presence of three terpenoids, out of which 1 terpenoid peak has wide area under curve that reveals the more availability of one terpenoid.

Invitro screening of anticataract activity of the hydroalcoholic leaves extract of Borreria hispida against galactose induction was studied. The photographic evaluation claimed that in galactose induced lens the opacity of lens is increased and the increased content of protein deposition noticed under microscope whereas in extract treated group there was no observation of opacity of lens and no deposition of protein in and around lens confirmed the anticataract property of the terpenoids present in it.

In the total protein assay, the total protein in normal control group was found to be 14.24+ 1.24, the galactose treated lens showed significant reduction of 5.26+0.64 mg/gm of protein, whereas in the extract treated group it was found to be 8.62+1.68 and in standard treated group the total protein was found to be 11.08+2.80. The reversal of the protein content compared to galactose induced group strengthen the anticataract activity of the hydroalcoholic leaves extract of Borreria hispida against galactose induction. This study is in agreement with the finding that the hydroalcoholic leaf extract of Borreria hispida has been significantly decreases the polyol levels by inhibiting the aldose reductase enzyme which converts galactose to galactitol, which is the prime cause of diabetic cataract.^[16] The exact mechanism involved behind it is yet to be confirmed, any how the tetracyclic triterpenoid presence in the plant may be responsible for this activity.

The extract was also found to reduce the protein aggregation. The literature supports that the extract of Borreria hispida has an effective antioxidant activity due to the presence of terpenoid content in it.

CONCLUSION:

In this study, the phytochemical screening, TLC and HPTLC analysis and the pharmacological studies -photocopy of the lens, protein content estimation proved the role of anticataract effect of the hydroalcoholic leaf extract of Borreria hispida against galactosamine induction. Further study reuired to Quantify the tetracyclic triterpenoid content present in it, finally isolation of phytomolecule is essential to formulate for the beneficiary of the society to prevent against blindness.

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Received on 12.11.2018 Modified on 15.12.2018

Research J. Pharm. and Tech. 2019; 12(3): 1235-1238.

DOI: 10.5958/0974-360X.2019.00205.1