Isolation of the natural colorant from the roots of Arnebia nobilis and its use as a new neutralization indicator

.Solution of natural dye from the roots of the plant Amebia nobilis in methanol is an excellent acid-alkali indicator capable of working in much wider range than the usual indicators phenolphthalein and methyl orange. Volumetric titrations of strong acid-strong alkali, strong acid-weak alkali and weak acid-strong alkali are performed and very satisfactory results found in the wider range of concentrations oo- 3 to 4 N) of acid and alkali. The structures of the indicator in acid and basic mediums are suggested. The pK of indicator is 5.21. The pH at the equivalence rnint in three types of titrations varies in the range of 3.75 to 11.70; Amax are found to be 235 and 250 nm. Active component, arnebin-7, is isolated pure and also used as indicator to confirm the results.

Amebia nobilis is a Boraginaceous plant and is a native of Afghanistan. The roots are the source of well known red medicinal dye. The chemical investigation of this medicinal plant was first reported in 1969 1 . When extracted with methanol, a dark maroon decoction is obtained which yields a red-black solid on the removal of methanol. Apart from the medicinal use 2 -4 and the food colorantS, the dye has different colours in acid and alkaline mediums. Certain natural colorants prove to be the better indicators 6 than the conventional indicators in use. We explored the possibility of its use as neutralization indicator for varied concentrations of acid and alkali. A comparative study with conventional indicators phenolphthalein and methyl orange has also been undertaken. Active component, arnebin-7, has been isolated and also used independently as indicator to confirm the results.

Results and discussion
Results of the three kinds oftitrations are given in Table   1.
Results of pH-metric titrations are given in Table 2 alongwith the A.max values of the dye at the equivalence points of acid-base titrations.
It is evident from the results that the present natural dye obtained from Arnebia nobilis is a better indicator in all respects to the popularly used phenolphthalein and methyl orange indicators for different type of neutralization titrations. It is also better in range. A successful titration of w-3 N alkali could be performed by the natural dye whereas this was not possible when phenolphthalein was used as an indicator. Moreover, while phenolphthalein fails in strong acid-weak base, and methyl orange fails in weak acid-strong base, this natural indicator is successful in strong acid-weak base as well as weak acid-strong base in most of tried ranges of titrations.
In the case of phenolphthalein in presence of dilute alkali, the lactone ring opens 7 and the triphenylcarbinol structure undergoes loss of water to produce the resonating ion, which is red. But in excess alkali the red colour disappears owing to the conversion to benzenoid structure. In the case of Amebia nobilis the active coloured ingredient is arnebin-7 which is comparable to phenolphthalein. We have isolated pure arnebin-7 also (confirmed by A.max 225 and 540 nm, IR (vmax) 1608, 1600, 1553 and 1447 cm-1 and 1 H NMR single proton triplet at 3.18 T, 4-proton multiplet at 7.40 T, 1-proton multiplet at 4.80 T, 2-proton singlet at 2.83 T and singlets at -2.55 and -2.40 T and m.p. 95°) using the method described by Shukla et al. 8 and used as indicator independently. The same results are obtained. This dye is red in quinonoid form but blue in other form.
It seems that a change from quinonoid form to benzenoid form in excess alkali takes place just like in phenolphthalein. In acidic medium arnebin exists in its quinonoid form (1). In alkaline medium the conversion to the form (Il) takes   The pK value of the present dye indicator is found to be 5.21.
place, responsible for blue colour.
The existence of two forms in acidic and alkaline mediums is confirmed by obtaining of two different "-max values at equivalence points-first by titrating the alkali with acid in burette and second by titrating the acid with alkali in burette.

Experimental
Isolation of dye : Authentic roots of Arnebia nobilis were procured from Dehradun. The dye was islolated from the roots of the plant as described earlier 9 . lOg of the crushed roots were kept in sufficient methanol in Soxhlet extractor for 24 h. Dark maroon extract with a brown tinge collected and fresh methanol added again and kept for 24 h. Procedure was repeated till colour of extract became light. All solutions were mixed and methanol was separated by vacuum distillation.
Isolation of arnebin-7: Active component was isolated pure as described by Shukla et at. 8 , and duly identified.
Indicator solution : The dye (1 g) was dissolved in methanol (100 ml) and its pH was determined (Systronics-324) and found to be 5.05. Amebin-7 (0.02 g) was dissolved in 2 ml methanol. Its pH was found to be 5.26.  7 . In each case, both the titrations (acid in burette and alkali in burette) were performed using present dye indicator and separately the phenolphthalein or methyl orange (2-3 drops). Strong acid with strong base, strong acid with weak base, and weak acid with strong base were titrated. The pH-metric titrations were also perfonned in each case. The reproducibility was better than ±3% .The pK value of the dye solution was determined using Henderson-Hasselbalch equations 7 . Similar titrations were performed using the indicator solution made of pure amebin-7. Same results were obtained. For the determination of "-max• a Systronics UV-Vis Spectrophotometer-liS was used.