RPF � III

PROFORMA FOR SUBMISSION OF FINAL REPORT OF RESEARCH PROJECTS

Part � 1: GENERAL INFORMATION

800 Project Code 8001 Institute Project Code No :Biochem I (813) 8002 ICAR Project Code No. : 206 Name of Institute and Division : Indian Institute of Spices Research, 801 P.B. No. 1701, Calicut, Kerala Name & Address of Institute : Crop Production & Post Harvest Technology 8011 8012 Name of Division/Section :Biochemistry 8013 Location of the Project : IISr,Calicut 8014 Project Title : Biogenesis of Pigments in Spice

Crops 802 Priority area :Biosynthesis 8031 Research Approach Applied Research Basic Research Process of Technology

Development Transfer of Technology

01 02 03 04 803 Specific area : Biochemistry 804 Duration of Project : 8041Duration of Project : 8042Date of start of project : May 1996 8043Likely date of completion of project : May 2004

805 Total cost/Expenditure incurred :25.5 lakhs

(Give reasons for variation, if any from original estimated cost) 806 Executive Summary : The present study mainly emphasis on the in vivo synthesis of the coluring pigment in the crop. As a preliminary step, the relative concentration of the other metabolites � essential oil,

oleoresin and phenolic acids as well as starch during plant growth were determined which

gives a basic information on the rate of biosynthesis of the compounds during rhizome

development and indicate the availability of other precursors required for biogenesis.

Three experiments where conducted using i) 14C-carbonate, ii) 2-14C-Malonyl CoA, iii)

1-14C-Phenylalanine.. The first experiment using 14C-carbonate gave a clue as to final

stage synthesis and speedy translocation of the pigments from the root to the rhizome.

Second experiment using 2-14C-Malonyl CoA, which is the intermediary precursor

resulted in the non significant incorporation of label in curcumin thereby ruling out the

2nd portable suggested by Roughling and Whiting. Third experiment was conducted to

check the probability of occurrence of the phenyl propanoid pathway suggested by

Giessman.

The first committed step in the biosynthesis of phenylpropanoids skeleton in higher plant

is the deamination of L-Phenylalanine to form t-cinnamate which is catalyzed by the

enzyme Phenylalanine Ammonia Lyase. The enzyme activity was monitored during

rhizome development which indicated higher levels during initial phase. Different

curcuminoids in various high and low variety / accessions could be correlated with PAL

levels. However, the percentage distribution of curcuminoid follows a general pattern

65:20:15 for curcumin I, II and III even though slight variation was observed between the

accession. Localisation of PAL in turmeric leaves using density gradient centrifugation

could reveal the presence of PAL in the microsomal fraction. Subfractionation of this

fraction confirms the distribution of PAL in the endoplasmic reticulum, proplastids and

plasma membrane

PAL could be purified from leaves 157-fold after ammonium sulphate fraction

followed by division exchange and gel filtration chromatography. The purified enzyme

subjected to electrophoresis gave single thick band corresponding to a molecular weight

of approximately 39,000 DA. This suggests the holoenzmyme to be a tetramer of similar

or identical subunits, in agreement with tetrameric nature of PAL in all organisms

examined to date.

807 Key words : Pigments, turmeric, curcumin,

precursors,enzymes, suspension cultures 808

Part � II: INVESTIGATOR PROFILE (Please identify clearly changes, if any in project personnel)

810 Principal Investigator : Name : Dr. (Mrs.) B. Chempakam 8102 Designation : Principal Scientist (Biochemistry) 8103 Division/Section : Crop Production & Post Harvest Technology 8104 Location : IISR, Calicut 8105 Institute Address : Indian Institute of Spices Research, P.B. No. 1701 Marikunnu (P.O), Calicut � 673 012, Kerala 811Co-Investigator 8111 Name : Dr. T. John Zachariah 8112 Designation : Senior Scientist (Biochemistry) 8113 Division/Section : Crop Production & Post Harvest Technology 8114 Location : IISR, Calicut 8115 Institute Address : Indian Institute of Spices Research, P.B. No. 1701 Marikunnu (P.O), Calicut � 673 012, Kerala

Part � III: TECHNICAL DETAILS 820 Introduction and Objectives : 8201 Project Objectives : Immediate objectives :

1. To assay the key enzymes involved in the biosynthesis of curcuminoids 2. To find out the precursors and site of biosynthesis 3. To establish the biosynthetic pathway of curcumin Long term objectives : To workout the biosynthetic pathway of curcuminoids in turmeric and to manipulate the pathway for in vitro production using cell cultures 8202 Background information and Importance of the project :\

Curcumin is the main colouring principle found in the rhizomes of Curcuma longa L. (Zingiberacea, Turmeric). The rhizomes usually contain 3-5% curcumin and two related compounds � demethoxy and bisdemethoxy curcumin. The pigment has a traditionally important role as colouring agent for food, cosmetics and textiles, apart from its recent role in biomedical sciences as anticancerous, anti-inflammatory, hypotensive etc. Biosynthesis describes the in vivo production of the compounds from the simpler ones and involves a number of enzyme systems and intermediates. In higher plants like turmeric, the pathway leading to curcumin may differ in detail from one site to another with respect to the starting materials used.

With the growing demand for natural dyes in the West, the market for turmeric and turmeric products is expanding. Further more, many countries have banned use of synthetic dyes in food and drugs. Hence if the pathways for the biosynthesis of the products are established, it is possible to enhance the production of the pigment by biochemical manipulation of the rate-determining steps. To achieve this end, the biochemical pathways are to be very clearly spelt out. So far, not much work has been done on the biogenesis of the pigment, except for the two pathways suggested � the Phenyl propanoid pathway (Geissman, 1969) and the acetate pathway (Roughly and Whiting, 1973). The confirmation of these pathways have not been worked out. Thus the two biosynthetic schemes mentioned are not fully accepted and further experimental evidence for these should be carried out. Hence the major objective of the scheme is to evolve a clear picture as to the biogenesis of curcumin, which is possible with the help of biochemical and tracer studies.

821 Project Technical Profile 1. Analysis of curcumin and other secondary metabolites (Essential oil and Oleoresin)

and starch during development in leaf, root and rhizomes. 2. Formation and distribution of curcuminoids in turmeric rhizomes 3. Assay of major enzymes involved in curcumin biosynthesis. 4. Cell fractionation studies on PAL 5. PAL activity during early germination phase in turmeric levels 6. Identification of phenolic acid precursors in turmeric. 7. Isolation and separation of curcuminoids in turmeric. 8. Light studies on PAL activity 9. Incorporation studies in turmeric seedlings using 14CO2

8211 Technical Programme : (Indicate briefly plan of procedure, techniques, instruments and special

materials, organisms, special environment etc.)

8212 Total man months involvement of component project workers:

822 Final Report on the Project:

(Detailed report containing all relevant data with summary of results (not exceeding 2-5 pages).

Materials and methods Five varieties including the four released varieties (Prabha, Prathibha, Suguna and Sudharsana) and other local variety (Allepy) were selected for the studies. The rhizomes were planted at the Institute campus during June 1999, following the normal agricultural and management practices. Samples (Leaf root and rhizome were taken starting from 120 DAS (Days After Sowing) upto 240 DAS, at 30 days intervals. The samples were dried and powdered and then subjected to analysis.

Curcumin

Curcumin was estimated colorimetrically following the ASTA method (ASTA, 1970). 100 mg powdered sample was refluxed with 30 ml alcohol for 1-2 hrs, filtered and made upto 100 ml with alcohol. The extract was then diluted 12.5 times and O.D read at 4.25nm and compared with a small standard curcumin solution.

Essential oil (E.O)

The dried powdered tissue (~30g) was hydro distilled for 3 hrs in a Clevenger apparatus. The volume of oil separated was noted. EO % = (Volume of Oil/Wt. of Tissue) x 100. Oleoresin (OR) OR was estimated using the cold � percolation method with acetone. 10g tissue was taken and extracted with acetone overnight. The pooled acetone extract of two elutions was evaporated to dryness and or determined gravimetrically. OR% = (Wt. of OR/Wt. of tissue) x 100 Starch 100mg tissue was washed with 80% alcohol thrice to remove the free sugar and digested with 52% perchloric acid thrice. The digest was pooled, made upto 100ml and suitable aliquots taken for starch estimation using Phenol-Sulfuric acid method, in terms of Glucose.

Gas Chromatographic analysis of EO from leaf, root and rhizome The volatile oils of leaf, root and rhizomes were collected as described above and 0.5 µl was injected to GC (Perkin Elmer, USA) with flame ionization detector. Nitrogen was used as the mobile phase, with a column temperature of 700 C- 2100 C, @ 50 C per minute. The column used was OV-17. Percentage of each component was determined from their area percent under corresponding retention time.

Results

1. Analysis of curcumin, Starch and other secondary metabolites (EO and OR) during plant growth in leaf, root and rhizome.

This item of work was taken up to see the level of pigments from the 3rd month after sowing in the different vegetative parts if turmeric and its relative variation with respect to other major components viz; EO, OR and Starch. Table 1-3 shows the percentage of curcumin and other constituents in developing rhizomes. A representative histogram (Var. Allepey) showing the changes during rhizome growth for all the constituents is shown in fig. 1. It is seen that generally between 120 and 150 DAS, maximum content of curcumin is seen in the five varieties which decreased gradually and remained stable after 180 DAS. Essential oil content (Table.1) decreases gradually as the rhizomes develop. Higher concentration was seen in the first two stages (120 and 150 DAS), where, among the varieties, higher concentrations were seen in Allepey and Prabha. Varieties, Suguna and Sudarsana were having only 4.4 to 4.8% in the early stages. The individual constituents of essential oil through Gas Chromatographic analysis is given in Table 2. The data show that in root and rhizome, the major constituents are ar-turmerone (46.8 & 31.5%) respectively, while in leaf, the major component is α phellandrene (32.62%). Oleoresin (Table 3) also showed a gradual decline during the maturity of the rhizome. The oleoresin in the early stages ranged from 17.5 � 24% in the immature rhizomes, while it ranged from 6.6 � 9.64% in the mature ones, accounting for about 50 � 70% of decline. Varieties Prabha and Allepey possessed higher OR (24.01% and 23.9 respectively) in the initial stages. As regards to starch (Table.4) a gradual was seen in all the five varieties during rhizome growth. Highest starch content was seen in Variety Prabha. Export for the variety Allepey, where as starch content increased by 8.7%, other varieties showed no change during the last two stages (210 DAS and 240 DAS). Higher dry recovery (DR) was seen in Varieties Prabha, Prathibha and Allepey (25.7%, 20.2% and 23.9% respectively) in the mature stage, while Suguna and Sudarsana had loser DR. (11.9% and 13.2% respectively). (Table.5)

In turmeric leaves also, Curcumin and EO content were higher in the initial stages, which declined with the growth of the plant. Interestingly, leaf EO was about 3 times higher in Suguna and Sudarsana, as compared to the other 3 varieties (Table 6). Curcumin content in these varieties showed as sudden decline, at later stages and was noticeably lower than the other three varieties. In roots, no definite pattern was seen with respect to Curcumin and EO (Table 7) but the oleoresin was maximum at 150 DAS and 180 DAS (12.4% - 16.8%) and came down at full maturity (7.33 to 9.8%). Among the varieties tried, roots from variety Allepey had highest curcumin content while Suguna and Sudarsana had highest EO and OR. The distribution of Curcumin with respect to the other major components showed that there occurs a steep rise in the secondary metabolites in the early stages i.e., upto 180 DAS, in the rhizomes. Oleoresin which constitute the fixed oils, non volatile oils, pigments, resins and carbohydrates was found to be 24 � 25% of the total weight of rhizomes. The fact that starch content was lower in the early stages suggests that free sugars will be easily available as precursors for the synthesis of secondary metabolites. The gradual increase in starch corresponds with a decrease in Oleoresin percentage on an average of 15%. It is seen that turmeric leaves are a good source of essential oil in the early stages and varieties Suguna and Sudharsana contains higher percentage and exhibited only slow decline during maturity. Both leaf and rhizomes had similar percentage. The individual components have to be estimated using GC � MS. The essential oil content in the roots of three varieties also showed a higher content in the early stages, which is almost double the quantity in other varieties. Curcumin levels did not show much variation among the leaves and roots, it remained stable upto the stage of maturity. These variations may either be due to the higher vegetative growth of varieties, Prathibha, Prabha and Allepey as compared to Suguana and Sudarsana. Gas chromatographic studies on individual constituents of essential oils from root, rhizome and leaf showed that ar-turmerone and ar-curcumene form the major components in rhizome and root oils. Leaf oil showed an entirely different pattern with α- phellandrene and Terpenolene as the major components, indicating a varied biosynthetic pathway for essential oils present in the leaves. The change in the component essential oils in these tissues during plant growth are being worked out. 2. Distribution of Curcuminoids in rhizomes. The absorption maxima of the curcuminoids along with its chemical formula are given in Table 8. The curcuminoids content as Curcumin I, II and III at different growth stages are seen in figs 1 a, b, c, d & e. The curcumin content in the pooled rhizome sample is given on Table 9. The results show that curcumin (curcumin I), which forms the major portion of the curcuminoids, is distributed uniformly in mother primary and secondary rhizomes only in the initial stage (120 DAS). As the rhizomes develop, the primary and secondary

rhizomes contain higher proportion of curcumin (61.8 � 73.6%) as compared to mother rhizomes (46.4%). At full maturity, however, primary rhizomes had the highest content. This indicates that at later stages there was a lower production of curcumin II and III, which are the two demethoxy forms. These compounds probably substantiate by providing the methyl acceptor to the final methylated form. Curcumin II (Demethoxycurcumin), which is structurally similar to Bis demethoxy curcumin (curcumin III), except for an additional methoxy group (-OCH3) was maximum in mother rhizomes at 150 DAS. The secondary rhizomes also possessed a higher level at full maturity. In all other stages, the compound was at low concentration and varied from 11-24 highest in mother rhizomes and lowest concentration was seen in secondary rhizomes. Eventhough the individual curcuminoids differ in their biological activity, there exists a synergestic functioning of the compounds. It is reported that the proportion of C, DC and BDC exists in a definite proportion in the rhizome, which can influence its bioprotective functioning (Majeed, 1995). Thus the �curcumin C3-complex� as it is rightly called, possesses a collective effect of all the curcuminoids. The present study was undertaken to observe the distribution of the three curcuminoids at various stages of plant growth so as to get a preliminary information on the in vivo synthesis of these compounds. This indicates that later stages there was a lower production of curcumin II and III, which are the two demethoxy forms. These compounds probably substantiate by providing the methyl acceptor to the final methylated form. The fact that the two demethoxy forms of curcumin (II & III) were seen at a lower level in the rhizomes, with a correspondingly high curcumin I indicates the conversion of the fully methylated form at the expense of these compounds. Table 9, which shows the curcuminoids in the pooled rhizome samples, exhibits a similar pattern, where maximum levels of curcumin III was observed at 180 DAS. The only difference was that DC and BDC are present in equal proportion, except at stage III where it was proportionately low as compared to Curcumin I. Assay of PAL during rhizome development The specific activity of PAL as µM of cinnamic acid is given in Table 10. Activity of PAL was very low in roots at 20 DAS, which gradually increased during the next samplings. In rhizomes and leaf, initial PAL activity showed an increase, which came down during the plant growth. In rhizomes, the activity decreased to about 1/5th of the initial value. In leaves also, a decrease of about 60% was seen after 80 DAS. PAL activity is depicted in table 10. There are several reports on the fluctuating levels of PAL activity in many annual crops w.r.t climate, soil, light intensity and variety. However, the activity was seen only upto 3 months after planting. The analysis will be continued upto full maturity of the rhizomes. Table 1. Essential oil (EO) percentage in turmeric rhizomes during development

Varieties Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Prabha 6.9 5.33 5.11 4.56 3.64 Prathibha 4.9 6.66 4.05 4.57 4.99 Allepey 7.2 6.33 4.1 4.39 5.3 Suguna 4.8 4.28 3.21 3.58 5.32 Sudarsana 4.4 4.28 3.02 2.59 5.24 Table 2. Major Essential oil constituents in turmeric rhizome, root and leaf (Through GC) Rhizome Root 600 Leaf Ar-turmerone (31.5) Turmerone (9.9)

Curlone (10.5) Ar-curcumene (6.27) β-sesquiphellandrene β-bisabolene (tr) Dehydrocurcumene (2.23) P-cymene (2.98)

Ar-turmerone (46.8) Ar-curcumene (7.02) B-sesquiphellandene (2.34) Dehydrocurcumene (4.27) P-cymene (3.3)

α-phellandrene (32.62) Terpenolne (25.95) P-cymene (5.0) 1,8-cineole (6.52) Myrcene (2.3) α-pinene (2.78) β pinene (2.13)

(Values in brackets show the percentage) Table 3. Oleoresin (OR) percentage in turmeric rhizomes during development Varieties Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Prabha 24.01 17.95 14.0 9.36 6.69 Prathibha 20.88 17.95 14.9 10.72 9.48 Allepey 23.96 19.38 18.9 11.96 10.72 Suguna 22.52 11.49 11.4 8.87 8.83 Sudarsana 17.55 15.42 12.6 9.09 9.64 Table 4. Starch (%) in turmeric rhizomes during development Varieties Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Prabha 39.6 42.3 47.8 54.2 56.1 Prathibha 38.5 39.9 42.9 60.1 59.4 Allepey 40.8 39.7 43.3 46.2 54.9 Suguna 39.6 42.7 39.6 52.1 51.7 Sudarsana 45.1 40.2 45.5 54.1 50.2

Table. 5 Dry recovery (%) of turmeric rhizomes during development Varieties Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Prabha 12.6 9.30 15.3 26.2 25.7 Prathibha 8.98 11.6 14.6 20.9 20.2 Allepey 8.10 9.30 14.1 19.5 23.9 Suguna 8.60 9.80 13.5 15.9 11.9 Sudarsana 8.40 8.20 9.7 13.2 13.2 Curcumin % Essential oil % Variety St.1 St.2 St.3 St.4 St.1 St.2 St.3 Prabha 0.196 0.167 0.159 0.15 2.2 1.6 1.12 Prathibha 0.167 0.157 0.164 0.123 2.6 1.9 0.99 Allepey 0.246 0.122 0.086 0.074 3.0 1.7 0.99 Suguna 0.203 0.112 0.082 0.027 4.4 3.3 2.92 Sudarsana 0.214 0.115 0.052 0.019 3.11 2.9 2.82 Table 7. Curcumin, Essential oil and Oleoresin in Turmeric roots during plant growth Curcumin Essential oil Oleoresin Variety St.1 St.2 St.3 St.4 St.5 St.1 St.2 St.3 St.4 St.5 St.1 St.2 St.3 St.4 St.5Prabha 0.9 0.9 0.7 1.1 0.6 4.5 4.3 3.5 4.6 4.9 8.0 12 9.6 10 7.3 Prathibha 0.9 1.3 1.2 1.4 0.7 5.3 5.6 4.4 3.0 4.3 8.6 14 10 11 7.8 Allepey 1.4 1.1 1.5 1.0 0.9 4.0 3.7 4.6 3.8 3.7 9.0 15 12 8.5 8.8 Suguna 0.9 1.1 1.4 0.9 0.7 8.4 12 5.8 8.1 5.0 10 17 13 11 8.5 Sudarsana 0.5 0.6 0.8 1.2 0.5 11 6.7 5.2 5.1 3.4 9.6 14 15 9.6 9.8 Table 8. Absorbtion maxima of curcuminoids Curcumin I Curcumin 428 Curcumin II Demethoxy curcumin 423 Curcumin III Bis Demethoxy Curcumin 418 Table 9. Total Curcuminoids in rhizomes during development Curcumin I Curcumin II Curcumin III

120 DAS 57.95 20.31 21.73 150 DAS 57.45 21.34 21.2 180 DAS 63.87 16.36 19.77 210 DAS 53.3 22.4 24.86 240 DAS 58.32 21.56 20.12 Table 10. PAL activity in turmeric during early stages of the plant growth (Expressed as uM of cinnamic acid released /min/mg protein) x 10-3 DAS Root Rhizome Leaf 20 6.87 822.6 302.9 40 81.07 204.5 224.8 60 218.6 212.5 70.05 80 283.5 144.2 120.01 DAS: Days After Sowing Turmeric rhizomes (Variety Prathibha) were sown in polybags. One month old seedlings of uniform size (30 nos.) were selected for the exposure studies. The seedlings were placed in a pepex glass chamber (1 x 0.5 m x 0.5 m) designed for the purpose. Labeled NaCO3 was placed in a petridish and placed in the center of the chamber and dil. HCL was added by drop through, a slit provided at the top. Addition of 1 ml of HCL can release 967 x 1017 14C-atoms which will be available for incorporation. Sampling: The chamber was sealed airtight after releasing the CO2 and kept intact for 24 hrs. Seedlings without exposure are taken as zero hr control. Seedlings were sampled at 24,48,72 and 96 hrs, one week for analysis. Afterwards wimples are being taken at 1-monthly intervals. Processing: Samples were processed for the extraction of phenolic acids and curcumin to see the incorporation of the labeled compound using Liquid Scintillation Counter. The analysis will be completed by February 2001. 4. Cell fractionation studies on PAL Five grams of turmeric leaf tissue was chopped and ground in 10 ml of borate buffer (0.1 M, pH 8.8). The extract was filtered through several layers of cheesecloth. The filtrate containing the suspension of cell components was centrifuged for 5 min at 200g to separate nuclei. The supernatant was again centrifuged for 10 min at 1000g to remove the residue containing chloroplast. The supernatant was then centrifuged for 15 min to remove the mitochondrial residue. Then the supernatant was centrifuged for 120 minutes at 1,05, 000g in an ultracentrifuge and the residue containing the microsomal fraction was removed. The residue in each case was dissolved in borate buffer and Phenylalanine ammonia lyase activity was estimated in all the fractions.

Cell fractionation of PAL activity Distribution of PAL in different cell fractionation of turmeric leaves showed maximum activity in mitochodrial fraction. (Table 1). Chloroplast and microsomal fractions were also rich in PAL activity. Table 11: PAL distribution in various cell fractions in turmeric leaves Speed Time Fractions

PAL activity (x 10-2)

200 g 1000 g 10,000g 1,05,000 g

5� 10� 15� 2 hrs

Crude extract R Chloroplast R Mitochondria R Microsomes R Soluble protein S

25.31 14.65 28.35 13.33 Nil

R � Residue S � Supernatant 5. PAL activity during early germination phase in turmeric leaves Turmeric rhizomes from two varieties (Prathibha and Suguna) were sown and samples were taken at 15-day intervals to assess the PAL activity for a period of 3 months. 5g tissue was extracted with Borate buffer pH 8.8 and the supernatant was taken for PAL assay. The activity was expressed as µM of cinnamic acid released/mg/protein. The activity was maximum at 15 DAS (Days After Sowing) in rhizomes which declined afterwards. (Fig1). The activity in roots showed as steady increase. 6. Identification of phenolic acid precursors in turmeric 5g of turmeric rhizome were digested with 20 ml of 2N HCL or 20 minutes. Filtered and extracted with 10 ml of diethyl ether thrice. The pooled ether layer again extracted with 5% Sodium Carbonate, thrice. Pooled the extract (sodium carbonate layer). Acidified this extract to pH 3 with 5% sulphuric acid. Extracted with 10 ml ether evaporated to dryness and dissolved in alcohol. The alcohol extract was applied to TLC.

TLC plates (Silica gel, thickness 1mm) were used for the separation of phenolic acids. Coumaric acid, Caffeic acid and Ferulic acid were used as standards and were spotted side by side with the sample. Solvent system used was Toluene: Acetic Acid in the ratio 4:1. The plate was run for about 1 hr and sprayed with 20% sodium carbonate followed by Folin�s Reagent. Blue colored spots appeared on the plate. Identification of phenolic precursors

The blue coloured spots corresponded to coumeric acid, caffeic acid and ferulic acid. Rf values of the three spots, which corresponded, with the known values are given below:

601 Phenolic acid

Rf values

Caffeic acid Coumaric acid Ferulic acid

5.2 7.9 9.73

7. Isolation and separation of curcuminoids in turmeric 50g of dried was powdered turmeric rhizomes was defatted with Hexane in a Soxhlet extractor and subsequently treated with methanol. The methanol extract was taken and the extract was then loaded into a silica gel column (3cm x 60 cm) which was pre-equilibrated with Petroleum Ether. The column was eluted with petroleum ether followed by a mixture of petroleum ether and ethyl alcohol prepared in the ratio 95:5, 90:10, 80:20, 60:40, 40:60, 20:80 followed by ethanol and finally by methanol.

On random application of these fractions to TLC plates, it was found that 60:40 fraction contained three distinct spots. Some additional faint spots were also obserced which might have come from the flavanoids co eluted curcuminoids. The three spots were identifies as curcumin I, II and III after comparing with standard curcuminoids. In another trial to separate curcuminoids, oleoresin from turmeric rhizomes was extracted using cold percolation in a column using acetone. Acetone was evaporated to dryness and a small amount of the oleoresin fraction was dissolved in 0.5 ml of alcohol and spotted on TLC plates of thickness 0.5 mm. A sample containing standard curcumin (sigma, USA) was also spotted alongwith the samples for comparison. TLC was run in a chamber equilibrates with the solvent system (95% methanol: 5% water) for 1 ½ hours. Three yellow fluorescent spots were seen on drying. The Rf value of each spot was calculated and compared with those of authentic standard of curcumin. Turmeric seeds were sown in poly bags and were allowed to germinate in darkness for 21 days. From this three sets were taken and were exposed to three sources of light viz. White, Red & Blue. The samples from each set where collected and the PAL activity was estimates at intervals of 30 minutes, 2hr, 4hr, 6hr, 8hr, 12hr and 24hr. A control was kept in dark and a control germinated in daylight was used as the light control. 8. Light studies on PAL activity

Exposure to red light shows peak activities at 6 hrs and 24 hrs, while blue light has peak activities at two different time periods (at 2 hrs and 8 hrs). White fluorescent light shows peak activity at later hrs i.e., after 6 hrs of exposure. Light control shows activity at later periods while dark control shows negligible activity at all time intervals.

The study suggests that in vivo synthesis of enzymes associated with phenolic acid metabolism is affected by light. The introduction of enzymes activity differs with time and between red and blue light. However noticeable changes in the activity are observed only at later hours of exposure, indicating the need for higher periods of exposure in turmeric to bring out significant changes. PAL activity in rhizomes, root and leaf at very early stages in turmeric (As µM trans-cinnamic acid released/min/mg/ protein x 10-2) Prabha Stages

I II III IV V

602 Rhizome

82.3 20.4 21.2 14.4 14.4

Root

6.8 8.11 21.8 25.38 28.4

Leaf

30.3 22.5 7.01 21.7 12.01

Suguna Rhizome

111.5 7.40 8.16 20.9 24.2

Root

3.38 3.71 5.32 10.61 9.44

Leaf

137.1 6.99 15.2 8.59 1.48

9.Tracer studies using 14C- CO3 14C � labeled potassium carbonate was purchased from BRIT (Board of Radiation Isotope Technology) for conducting the incorporation studies of 14C in the very early stages of rhizome growth (upto 3 months) with regard to phenolic acids, acetate and other components. For this, a dummy experiment was conducted using Na2CO3. 12 seedlings were placed in a Perspex glass chamber (1 mx 0.5 mx 0.5 m), designed for this purpose. Na2CO3 was placed in this chamber and dil HCL was added slowly so as to evolve CO2. The plants after exposure to 30 min. were replanted in pots. Samples were taken for standardizing methods for extraction and estimation of phenolic acids and separation through TLC to identify the individual phenolic acids.

Table 12.Incorporation of 14C Phenylalanine in turmeric plants

Control Test Period Tissue Wt (g) DPM Wt (g) DPM

0 hr Leaf Pseudostem Rhizome Root

1.05 1.00 0.49 0.07

14.6 1.9 9.7 2.1

48 hr Leaf Pseudostem Rhizome Root

0.50 0.45 0.11 0.22

9.9 7.2 2.2 8.0

1.06 0.28 0.06 0.03

71.2 70.0 38.4

4968.4

1W Leaf Pseudostem Rhizome Root

1.02 0.05 0.29 0.99

21.0 0.7 0.0

10.1

1.29 1.72 1.34 0.64

114.9 102.7 243.9

1987.6

1M Leaf Pseudostem Rhizome Root

2.05 1.50 3.40 1.42

0.2 4.7

12.1 2.1

3.15 2.00 1.05 0.98

105.9 44.3 63.6

983.7

Table 13. Incorporation of 14 C-CO2 as DPM (Disintegration per minute) in phenolic acids of turmeric leaves Period DMP Sample quality

measurement 24 hrs 7483.0 4.8 7478.2 0 0 48 hrs 19329.6 32.2 19297.4 0 0 96 hrs 22432.2 11.6 22420.6 0 0 1 Week 10866.6 23.6 10843.0 0 0 1 month 1259.6 54.2 1205.4 999 0 2 month 14.1 12.6 1.5 0 0 3 month 27.0 12.8 14.2 0 0 4 month 35.5 0.0 35.5 999 0 5 month 29.2 1.9 27.3 0 0 6 month - - - - -

Table 14. Incorporation of 14 C-CO2 as DPM (Disintegration per minute) in phenolic acids of turmeric rhizomes Period DPM Sample quality

measurement 24 hrs 11219.6 12.6 11207.0 0 0 48 hrs 11953.9 26.3 11927.6 0 0 96 hrs 11439.7 21.9 11417.8 0 0 1 Week 8508.9 8.8 8500.1 0 0 1 month 962.5 13.8 948.7 0 999 2 month 2.0 16.4 -14.4 0 0

3 month 35.4 1.2 34.2 0 0 4 month 73.5 12.9 60.6 0 0 5 month 55.7 0.0 55.7 0 0 6 month 9.4 0.0 9.4 0 0

Table 15. Incorporation of 14 C-CO2 as DPM (Disintegration per minute) in phenolic acids of turmeric pseudostem

Period DMP Sample quality measurement

24 hrs 15151.9 14.9 15137.0 0 0 48 hrs 3333.0 0.0 3333.0 0 0 96 hrs 5727.4 9.3 5718.1 0 0 1 Week 12648.9 48.4 12600.5 0 0 1 month 580.9 21.3 559.6 999 0 2 month 13.3 0.0 13.3 0 0 3 month 17.8 2.4 15.4 0 0 4 month 1.1 -1.1 0 5 month 20.1 17.2 2.9 0 0 6 month 173.0 37.2 135.8 0 0

Table 16 Incorporation of 14 C-CO2 as DPM (Disintegration per minute) in phenolic acids from turmeric roots Period DPM Sample quality

measurement 24 hrs 88.4 24.6 63.8 0 0 48 hrs 9908.3 10.9 9897.4 0 0 96 hrs 2238.7 27.6 2211.1 0 0 1 Week 4895.2 14.3 4880.9 0 0 1 month 1109.4 19.5 1089.9 999 0 2 month 0.0 12.2 -12.2 0 0 3 month 42.8 8.8 34.0 0 999 4 month 60.1 1.1 59.0 0 0 5 month 407.2 9.3 397.9 0 0 6 month 16.7 4.5 12.2 0 0

Table 17. Incorporation of 14 C-CO2 as DPM in various vegetative parts of turmeric during rhizome development Period Root

(T-C) Rhizome

(T-C) Pseudostem (T-

C) Leaf (T-C)

24 hrs 63.8 11207.0 15137.0 7478.2 48 hrs 9897.4 11927.6 3333.0 19297.4

96 hrs 2211.1 11417.8 5718.1 22420.6 1 Week 4880.9 8500.1 12600.5 10843.0 1 month 1089.9 948.1 559.6 1205.4 2 month -12.2 -14.4 13.3 1.5 3 month 34.0 34.2 15.4 14.2 4 month 59.0 60.6 -1.1 35.5 5 month 397.9 55.7 2.9 27.3 6 month 12.2 9.4 135.8 -

Fig.1

Subcellular localisation of Phenylalanine Ammonia Lyase Subcellular localisation of Phenylalanine Ammonia Lyase (PAL, E C. 4. 3. 1. 5), which is

the key enzyme in the Phenyl propanoid pathway, was investigated in the leaves of

turmeric (Curcuma longa L.). Cell fractionation carried out using differential

centrifugation with sucrose gradient indicated the presence of the enzyme in the

microsomal fraction. Further subfractionation of the microsomes indicated the presence

of PAL in Endoplasmic Reticulum (ER), proplastids and plasma membrane. However

maximum activity for PAL was seen in ER. This is in conformity with the findings that

ER is a site for phenyl propanoid and flavanol metabolism.

Curcumin,Essential Oil,Oleoresin,Starch and Dry recovery in turmericrhizomes during development( Var. Alleppy)

0

10

20

30

40

50

60

Curcumin(%)

EO (%) OR(%) Starch(%) DR(%)

120 DAS150 DAS180DAS210 DAS240 DAS

Purification of PAL and properties

L-phenylalanine ammonia lyase (PAL, E. C. 4. 3. 1. 5) has been purified from Curcuma

longa L. leaves. After preliminary purification by ammonium Sulphate fractionation and

DEAE- Sephacel ion exchange chromatography, the enzyme was further purified using

Sephacryl S-300- gel chromatography. The purified enzyme preparation has

homogeneous subunits, which exhibited a Mr ranging 38,0000- 39,0000.Optimum pH,

temperature and substrate concentration

Effect of light on PAL activity

Etiolated turmeric seedlings were exposed to red, blue and white light to study the

induction of PAL activity. Exposure time was 72 hrs and sampling was done at 6 hr

intervals. Exposure of red light and white light gave two peak activities at 18 and 48 hrs.

with a lag phase of 12 hrs in the case of red light. However white light gave higher

extractable PAL activity causing a lag phase of 6 hrs. Blue light also gave a single, but

not so predominant peak at 12 hrs. A characteristic feature of these light induced

increase in the enzyme activity is the lag phase of 12hrs, indicative of a phytochrome

mediated response. In short, exposure of turmeric seedlings to the three forms of light

evokes varied response to PAL, possibily via de novo synthesis or through a derepression

factor or through feed back inhibition.

8221 Achievements in terms of targets fixed for each activity : 8222 Questions � Answered: PAL has been confirmed as the rate limiting enzyme in curcumin biosynthesis

8223 Process/Product/Technology/Developed : Nil 8224 Practical Utility:

(not more than 150 words)

Curcumin, which belongs to the natural diarylheptanoids is the major pigment present in the rhizomes of Curcuma longa L. (turmeric) and other Curcuma spp. The rhizomes contain three yellow pigments � Curcumin I, Curcumin II (Demethoxycurcumin) and Curcumin III (Bis demethoxy curcumin). Today, turmeric, turmeric extracts and oleoresins are commercial products produced in large quantities used as colouring matter in food processing around the world. Apart from its industrial uses, curcumin has got a broad spectrum of medicinal and pharmacological properties and is regarded as a drug/drug model. Thus, in the indigenous systems of medicine in the orient, turmeric has been used since time immemorial. It enjoys the reputation as an antiinflammatoric agent, as a carminative, diuretic and blood purifier as well as a remedy against jaundice. It is also recommended for use against common cold, leprosy, fever, liver infections and also in the treatment of ulcers. In fact, curcuminoids I, II and III are found to be the most potent anticarcinogens, inhibiting mutagenesis and carcinogenesis. The effect might be due to its ability to scavenge oxides and peroxides .t has also been identified as the most recent anticancer drugs in Chinese literature .. The anti-ulcerogenic and wound healing effect of curcumin is explained based on its inhibitory effect on thromboxane-2 release. . In short, during the last ten years, new attention has been given to turmeric and the pharmacological studies on isolated curcumin. The blood sugar lowering effect of curcumin (Sreenivasan, 1972), its effect on the mucin content of gastric juice (Gupta et al, 1980; Prasad et al. 1976) and its anti-rheumatic of RES activating property have also been reported (Gondo et al, 1996).

!Choleretic ! Antiheppatotoxic ! Cytotoxic ! Hypocholesteremic ! Antiinflammatory ! Blood sugar lowering ! Anti-rheumatic ! Spasmolytic ! antibacterial ! Hypotensive

With this background information on hand, it seems worthwhile to find out how the pigment is being synthesized in the plant so that the information arising out of the project can provide valuable tools for further genetic and biotechnological studies. Very little work has been carried out for establishing the biosynthetic pathway of curcumin except for the two schemes proposed by Geissman (1969) and by roughly and Whiting (1973). Thus, identification of the pathway can ultimately give a lead in the following aspects:

a) Locating the site of synthesis of curcumin in turmeric b) To elucidate analogous pathways of biogenesis of secondary metabolites in other

spice crops (black pepper, ginger etc.).

c) To establish protocols for developing cell lines capable of over production of the pigment. This can be achieved by manipulating the biochemical pathways in vitro.

8225 Constraints, if any: Nil 823 Publications and Materials Development:

(One copy each to be supplied with this proforma.)

8231 Research papers: Chempakam,B, Zachariah TJ, K Kandiannan 1999 Activity of PAL and other secondary metabolites during rhizome development in turmeric : Paper presented in the National Symposium on Plant Physiology and Biochemistry, DKVV, Indore,Feb 15-17,1999. Chempakam, Leela, NK, Sinu P John 2000 Distribution of curcuminoids in turmeric during rhizome development. Paper presented during the Centennial Conference on Spices, Medicinal and Aromatic Plants, Sep 20-23,2000 (Awarded as the best poster paper during the Centennial Conference on Spices, Medicinal and Aromatic plants, 20�23 September 2000) Leela, NK, Aldo Tava, Shaji PM, Sinu P John and Chempakam B (2002) Composition of

essential oils from turmeric (Curcuma longa L), Acta pharmaceutica, 52:137-141

Neema Antony, B.Chempakam and A. I. Bhat (2005) Subcellular Localisation of PAL in

Curcuma longa L ( Communicated.)

Neema Antony, B.Chempakam (2005) L- phenylalanine ammonia lyase from Turmeric- purification and properties (Communicated)

Neema Antony, B.Chempakam, Sinu John, K.Vasu, NK Leela, Shahul Hameed, and S. Sindhu (2006) Tracer studies on the biosynthesis of curcumin in turmeric (Curcuma longa L.) using 14C-CO2, 1-14C-phenyl alanine and 2-14C-Malonyl Co A ( Comunicated )

8232 Popular articles: Nil

8233 Reports:

8234 Seminars and workshops (relevant to the project) in which the Scientists have participated (List abstracts forwarded):

National Symposium on Plant Physiology and Biochemistry, DKVV, Indore,Feb 15-17,1999.

Centennial Conference on Spices, Medicinal and Aromatic plants, 20�23 September 2000

824 Infrastructural facilities developed:

HPLC system installed for analysis of curcuminoids Field Note books- 5 nos

825 Comments/suggestions of Project Leader regarding possible future line of work that may be taken up arising out of this Project:

Phenyl alanine ammonia lyase has been found to be the rate limiting enzyme in curcumin biosynthesis. The activity has been correlated with the high and low curcumin accessions. It is worthwhile if programmes on cloning and sequencing of the enzyme and identifying the gene encoding PAL are taken up. Meanwhile the activity of other downstream enzymes in the pathway can also be worked out to find out more control points.

Part � IV: PROJECT EXPENDITURE (Summary)

Year�.

830 Total Recurring Expenditure:

8301 Salaries: (Designation with pay scale): Estimated Actual i). Scientific : 13.440 15.36 ii). Technical : 1.696 1.865 iii) Supporting: : 0.672 0.739 iv). Wages: : Sub Total : 15.808 17.964 8302 Consumables: i). Chemicals : 4.0 6.0 ii). Glasswares : 1.0 1.0 iii). Others : 1.0 1.0 Sub Total : 6.0 8.0 8303 Travel : Nil 8304 Miscellaneous : 1.50 2.50

(other costs)

8305 Sub total (Recurring) : 23.808 28.464

831 Total Non-Recurring Expenditure

(Equipments and works) i).HPLC accessories 2.0 5.0 ii). iii).

832 Total (830 & 831) : 25.308 33.464

Part-V: DECLARATION This is to certify that the final report of the Project has been submitted in full consultation with the Project workers as per the approved objectives and technical programme and the relevant records, note-books, materials are available for the same. Signature of the Project Investigator Co-Investigators:

1. 2. 3. Signature and comments of the head of the Division/Section : Signature and comments of the Joint Director (Research) : Signature and comments of the Director :