QUANTITATIVE DETERMINATION OF RUBIADIN IN DIFFERENT ACCESSIONS OF RUBIA CORDIFOLIA LINN. BY ISOCRATIC RP-HPLC

1. Department of Biotechnology, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi-110062, India. 2. Department of Botany, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi-110062, India. 3. Department of Botany, Aligarh Muslim University, Aligarh-202001, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Rubia cordifolia L. (common name-Indian Madder, Majith, Manjistha) from the family Rubiaceae is commonly known as is widely dispersed throughout the lower hills of Indian Himalayas (Shekhar et al ., 2010;Radha et al., 2011). The plant is important and famous drug in the Ayurvedic treatments. Extracts of this plant have shown hepatoprotective, antineoplastic properties and is also useful in the disintegration and elimination of urinary stones (Gilani and Janbaz,1995;Divakar et al., 2010). Anti-inflammatory, anti-ulcer and anti-dysentricactivities are also found in the roots of Rubia cordifolia Linn. (Deoda et al., 2011). It is also used in the treatment of diuretic, liver complaints, joint pains, uterine pains, in rheumatoid arthritis (Shekhar et al., 2010).

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Although, HPLC method for determination of rubiadin in the roots of Rubia cordifolia Linn. has been reported in literature. But quantitative determination of rubiadin in different accessions of the plant has not been reported yet. Further we also quantified rubiadin in both roots and aerial parts of Rubia cordifolia Linn. which has also not been reported yet. Present study deals with the quantitative determination of rubiadin by RP-HPLC in methanolic extracts of root and aerial parts of the different accessions of the plant. Briefly, in this study we verified the RP-HPLC method for the quantification of the rubiadin in different accessions of the plant.

Material and Methods:-Plant material:-
Six different accessions of Rubia cordifolia Linn.were used in this study. Roots and aerial parts of plant were collected from six different geographical areas (Table. 1) of India. These samples were identified by Prof. M. P. Sharma, Head, Dept. of Botany, Jamia Hamdard. Voucher specimen is deposited at the herbarium of Department of Botany, Jamia Hamdard. New Delhi.

Extraction of Plant material:-
The plant samples were air dried and ground into a fine powder using a grinder. Then, the plant material was extracted using different solvent system in a soxhlet apparatus and by other methods as well. 20g dried plant material was extracted using 200 ml of each solvent system of increasing polarity like petroleum ether, chloroform, acetone, ethanol (80%) , methanol ethyl acetate, n-butanol and water respectively in a soxhlet assembly for 12 h. Each extract was concentrated by distilling off the solvent and then evaporating to dryness on water bath or using Rotary evaporator. Extracts were also used directly for various tests. Extracts were collected and used for different phytochemical tests (Results in Table. 2) HPLC chemicals and reagents:-HPLC grade standard compound rubiadin was purchased from Natural Remedies (India). All the solvents and reagents used in the experiments were of HPLC grade. HPLC grade methanol and water were purchased from Merck, India. HPLC analysis was carried out on a Waters HPLC system (Binary Pump 600 controller), Waters PDA detector (996) and an auto sampler (2707). Empower 2 software was used to control the system and for monitoring and analysis of results. For chromatographic separation R p C 18 column (250×4.6 mm, particle size 5 µm) was used. Further, a sonicator, rotary evaporator (R-200/205/V (Buchi), a pH meter and hot air oven were also used. rate was 1 mL/min. The solvent was filtered through a nylon membrane (0.45µm) and degassed by sonication before use. UV spectra was recorded from 210-400nm at a rate of 1.00 spectrum/sec and a resolution of 1.2 nm.

Preparation of sample solution for HPLC analysis:-
Calibration:-Standard solutions of 10-200 µg/mL of rubiadin were prepared in methanol from the stock solution of 1 mg/mL and were used for the preparation of calibration graph. 20 µL of each of the standard solution was injected by the auto sampler with concentrations mentioned above and the linearity of response for rubiadin was determined. Calibration curve was drawn by plotting the peak areas rubiadin against the corresponding concentration.

Results & Discussion:-
The separation of rubiadin by RP-HPLC was carried out under optimized conditions. Optimization of mobile phase was carried out using various concentrations of methanol and water. Three different compositions of methanol and water were used, 75:25, 85:15 and 80:20. The optimum mobile phase was found to be Methanol: Water (HPLC grade) in the ratio of 80:20 it is the same as reported in the Khodke et al, 2010 method. Retention time of rubiadin was found to be 7.920 min as shown in Fig.1.A linear relationship between peak areas and concentrations was obtained in the range of 10-50μg/ml. This shows that method is linear. Repeatability studies show %RSD to be less than 2%.This shows that method is precise. %RSD for inter-day precision was higher than that of intra-day precision. Excellent recoveries were obtained at each level of added concentration as the mean recovery found to be within 98% to 102% for rubiadin. The limit of detection and limit of Quantitation of method was found to be 55.75ng/ml and 200ng/ml (Table.3). As it was found that rubiadin peak gets well resolved from peaks of other chemical constituents, hence we conclude that method is selective. Further, the quantification of rubiadin in different samples of R. cordifolia was also successfully done ( Fig. 2; Table. 4) Quantification of rubiadin in roots of R. cordifolia has been reported in the previous study (Khodke et al., 2010). This analysis showed that rubiadin is present in roots only but in our study we quantified the rubiadin in both roots and aerial parts of this plant. This study not only supports the previous study that roots are good source of rubiadin but also revealed that significant amount of rubiadin is also present in the aerial part of this plant. Thus, the whole plant can be utilized to extract rubiadin. Further, we also quantified the rubiadin in aerial and roots of the R.cordifoliacollected from different geographical areas which has not been reported earlier. Among all the six accession that has been used in the study the accession no.5 has the higher amount of rubiadin in its roots.