INTRODUCTION:

Rubia cordifolia Linn. is a flowering plant species. It is commonly known as Manjistha. Roots and stems are active part of plant. Rubia cordifolia Linn has many pharmacological actions like blood purifier activity, anticancer, astringent, antidysentric, antiseptic, deobstruent properties, antirheumatic^{1, 2}. Rubia cordifolia Linn contains various chemical constituents like Anthraquinones³, Iridoids⁴, Hexapeptides, Rubiprasins, Quinones, Triterpenoids⁵. Literature survey reveals that Quality control protocol has been developed for stems of Rubia cordifolia Linn⁶. There are number of HPLC and HPTLC methods were developed for chemical constituents of stem and root of Rubia cordifolia Linn e.g. HPLC methods was developed for Hexapeptide from roots of Rubia cordifolia Linn⁷, a gradient HPLC method for Rubiadin from stem of Rubia cordifolia Linn⁸ and HPTLC methods were developed for Purpurin⁹ and Quinonoids¹⁰ from the roots of Rubia cordifolia Linn. But to the best of our knowledge there is no HPLC method reported for this isolated anthraquinone marker. As it contain anthraquinone nucleus, it may have various activities like hepatoprotective, antioxidant. The aim of the present study accordingly was to develop RP-HPLC method for anthraquinone marker and validate it as per ICH guideline¹¹

MATERIALS AND METHOD:

Powder of roots of Rubia cordifolia Linn. were purchased from Total Herb Solution firm, Mumbai. It was authenticated by Botanical Survey of India, Pune. Chemical used for the experimentation was of laboratory reagent (LR) grade and analytical reagent (AR) grade. Chemicals used for this experiment were Methanol, Acetone, Ethyl acetate, Dichloromethane, Toluene, and Double Distilled Water.

Instrumentation:

Instruments used during research work were HPLC (Make JASCO) with UV detector, UV spectrophotometer (Make JASCO), Rotary Evaporator, Electronic Balance (Make SHIMDZU Model AY-120). Isolation of Marker was done by making modification in procedure reported in research paper by Singh R and Geetanjali¹². RP-HPLC Chromatographic method was developed using column Hi-Qsil C₁₈(250 × 4.6 mm) using UV detector (Model 2050).

Isolation of Marker:

Acetone: water extraction:

The air dried roots of Rubia cordifolia were percolated three times in acetone: water (1:1) for 48Hrs at room temperature. The combined decanted solvent mixture was distilled by simple distillation to remove acetone. A brown colour solid separated after removal of acetone. Then this brown colour solid was mixed with Dichloromethane for 15 min.

Column chromatography:

Preparation of sample:

Dichloromethane fraction was mixed with silica gel 60-120 mesh for column chromatography.

Preparation of Column:

Silica gel (60-120 mesh) was dried by keeping it in oven. Column was packed using this dried silica gel. Prepared sample was placed over that and again packed silica gel was packed on it.

Elution of Column:

Column was eluted with Toluene: Ethyl acetate in the ratio of 6:1. Every 2ml of eluent was collected in separated in test tube.

Thin Layer Chromatography:

Spotted the solution of each test tube over TLC plate pre-coated with silica gel 60 F₂₅₄ (10 cm ×10 cm with 250 mm layer thickness) and developed using mobile phase toluene: Ethyl acetate in the ratio of 6:1. Then on the basis of result obtained, the test tube which showed fluorescent compound at Rf 0.80, was selected for further processing.

Marker isolation:

All test tube solution was collected containing single fluorescent compound. Evaporated all liquid solvent and collected solid compound.

Preparation of standard stock solution:

a) Standard stock solution:

2mg of marker was accurately weighed and dissolved in 10 ml of chloroform to obtain stock solution of marker (200µg/ml).

b) Working standard solution:

0.1 ml of standard stock solution was diluted to 10ml with methanol.

Selection of Detection Wavelength:

Scanned the solution of Marker over range 400nm to 200nm. From the UV spectrum of Marker, selected the wavelength having maximum absorbance i.e. the wavelength was found to be 222nm.

Method development:

Selection of Mobile phase and chromatographic condition:

Chromatographic studies were carried on working standard solution using C₁₈ column. Trial for optimum mobile phase started with methanol and water (HPLC grade) in proportion of 1:1. After several trials most suitable mobile phase was found to be Methanol and double distilled water in the ratio of 80:20. This mobile phase gave peak with acceptable retention time and acceptable peak parameters.

Summary of chromatographic parameters selected:

a) Column : HiQ-SiL C₁₈ column (250 x 4.6 mm i.d.)

b) Mobile phase : Methanol: water (80:20 %v/v)

c) Flow rate : 1.00 ml/min

d) Detection : 222 nm

Wavelength

e) Sample injector : 50 µl loop

f) Temperature : ambient

Method Validation:

Validation of method was performed by using parameter as per ICH guideline.

Linearity:

Linearity of the method was checked using five different concentrations of marker. Injections of 200ng/ml, 400ng/ml, 600ng/ml, 800ng/ml, 1000ng/ml of marker was given. Then find the relation between concentration and area under curve was determined.

Precision:

Inter-day and Intra-day precision values were evaluated by analyzing standard preparations, six times and % RSD value was calculated to determine any intra-day and Inter-day variation.

Accuracy:

To check accuracy of the method, recovery studies were carried out by addition of standard drug solution to sample solution at three different levels 80, 100 and 120 %. Mean percentage recovery was determined.

Limit of detection and limit of quantitation:

The limit of detection (LOD) and limit of quantitation (LOQ) were obtained by calculating using the standard formula as per the ICH guidelines,

LOD = 3.3 σ LOQ = 10 σ

S S

Where σ is Standard deviation of the response and S is slope of the calibration curve.

Selectivity

As the method detected the marker in presence of other chemical constituent, hence method considered as selective.

Quantitation of marker:

Preparation of Sample solution:

Extraction was carried out as per the procedure given in Ayurvedic Pharmacopoeia.

2gm root powder of Rubia cordifolia was accurately weighed and dispersed in 20 ml of Dichloromethane. This solution was kept aside overnight at ambient temperature. The solution was filtered through Whatman filter paper No. 41 and filtrate was evaporated on a water bath to obtain a solid mass of extract. This extract was used for further quantitation (yield 0.9 %). The extract was dissolved in methanol and diluted appropriately. Then run the chromatogram of this solution for 15min.

RESULTS:

Development of the optimum mobile phase:

The optimum mobile phase was found to be Methanol: Water (HPLC grade) in the ratio of 80: 20. Retention time of Marker was found to be 4.233 as shown in Fig.1.

Table 1: Validation Summary of marker

Sr. No.	Validation Parameter	Marker
1	Linearity(r²)	0.9987
2	Regression Equation	y = 603.43 + 9021.1
3	Range	200 ng/ml to 1000ng/ml
4	Precision	1.8545
5	Accuracy	98-102%
6	LOD	25.21 ng/ml
7	LOQ	83.16 ng/ml
8	Specificity	specific

Table 2: Quantitative determination of marker

Supplier

Peak area

%RSD

Amount Marker

(mg/gm)

Mean

THS firm

297437

297889

296798

297474

548.16

0.19

0.936

Fig.1: Chromatograms of marker having RT 4.235 Having concentration 600 ng/ml.

Fig. 2 Linearity of marker

Fig.3 Chromatogram showing presence of marker in mixture of compounds isolated from roots of Rubia cordifolia Linn.

Validation of the developed method:

The validation summary is given in Table 1.

Quantitative determination of Marker as shown in table 2

Linearity shown in Fig.2

Chromatograph for extract as shown in Fig.3

DISCUSSION

This paper can be used for isolation procedure for isolation of anthraquinone marker and RP-HPLC as a quantitative determination method for anthraquinone marker.

REFERENCES:

1) Kamboj VP, Herbal Medicines. Current Science. 2000; 78: 35-39.

2) www.divineremidies.com accesed on 11^th AUG 2009.

3) Akhtar MS, Ali M, Madhurima, Mir SR and Onkar S. New anthraquinones from Rubia cordifolia roots. Indian Journal of Chemistry. 2006; 45B: 1945-1950.

4) Kuruuzum-UZ Ae, Guvenalp Z, Demirezer LO, Berger I and Strotch K. 4’ deoxy iridoids glycosides from Centranthus longiflorus. 2002; 61(8): 937-941.

5) Mishchenko NM et al. Chemical composition and pharmacological activity of anthraquinones from Rubia cordifolia cell culture. Pharmaceutical Chemistry Journal.2007; 41(11): 38-41.

6) The Ayurvedic Pharmacopoeia of India, Government of India Ministry of Health and Family Welfare Dept. Of Indian system of Medicine and Homeopathy New Delhi. 1^st edition, Volume 1(1), 2001,--

7) Lee JE, Hitotsuyanagi Y, Kim IH, Hasuda T and Takeya K. A novel bicyclic hexapeptide, RA-XVIII, from Rubia cordifolia: Structure, semi-synthesis, and cytotoxicity. Bioorganic and Medicinal chemistry letters. 2006; 18(2): 808-811.

8) Indian Pharmacopoeia. Volume ɪɪɪ, New Delhi, The Controller of Publication, 2007, 2049.

9) Thakur AK, and Hamrapurkar PD. Quantitative densitometric HPTLC analysis of purpurin in parts of Rubia cordifolia Linn. and in pharmaceutical dosage form. Journal of Planar Chromatography. 2008; 22: 109-113.

10) Hazra B, Sarma MD and Sanyal U. Separation method of Quinonoid constituents of plants used in oriental traditional medicines. Journal of Chromatography B. 2004; 812(1-2): 259-275.

11) ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2 (R1), Nov 2005.

12) Singh R. and Geetanjali. Isolation and synthesis of anthraquinones and related compounds of Rubia cordifolia. 2005, 70(7), 937-942.

Received on 18.08.2010 Modified on 01.09.2010

Research J. Pharm. and Tech. 4(2): February 2011; Page 326-328