Indian Journal of Natural Products and Resources Vol. 4(4), December 2013, pp. 398-411

Phytochemical studies of gomezianus Wall. ex Trecul. var. lakoocha Roxb. fruits collected from various altitudes of Central Western Ghats

S R Krishnamurthy* and P Sarala Department of Applied Botany, Kuvempu University, Jnana Sahyadri, Shankaraghatta – 577 451, Shimoga District, Karnataka,

Received 29 November 2012; Accepted 13 June 2013

The fruits of Artocarpus gomezianus Wall. ex Trecul. var. lakoocha Roxb. were collected from different altitudes of Central Western Ghats which differ in their topography, vegetation, social, cultural and food habits of the local people. The screenings of secondary metabolites revealed presence of alkaloids, phenols, flavonoids, tannins, steroids and saponins in the fruit samples of all the regions. The quantitative determination of secondary metabolites shown that the alkaloids were dominant at all the sampling stations followed by lignin, phenols, flavonoids, tannins and steroids. However, the amount of steroids and tannins were same at all the sampling stations except at higher altitudes where tannins are least. There was a shifting among the flavonoids and phenols in the middle lower and middle higher altitudes. Though alkaloids were dominant secondary metabolites and steroids were the lowest, with respect to concentrations of individual alkaloids, remarkable variations were observed. Among the six secondary metabolites, the concentration of steroids, tannins and phenols were high at the lower and higher altitudes and low in middle altitudes. In contrast to phenols, tannins and steroids, the concentration of flavonoids was more in the middle and lower in higher altitudes and lowest in higher and lower altitudes. The concentration of alkaloids and lignin did not show any relationship with variation in altitudes. The quantitative values of secondary metabolites are subjected for correction studies with reference to different altitudes, average rainfall and temperature. The +ve and –ve correction at 0.01 and 0.05 level is emphasized. The simple correlation coefficient between weather data and secondary metabolites revealed significant information. The percentage of secondary metabolites showed P ≤ 0.01 level significance with altitudes at all the stations except a few sites. The qualitative separation of secondary metabolites by thin layer chromatography method revealed that alkaloids, flavonoids and glycosides have three distinct spots with different hRf values. Steroids revealed two spots and saponins revealed one spots along with different hRf values.

Keywords: Artocarpus gomezianus, Central Western Ghats, Phytochemicals, Under-utilized .

IPC code; Int. cl. (2011.01)−A61K 36/00.

Introduction metabolites like tannins, saponins, photobatalins, The 75- 80% of the world population depends on flavonoids, terpenoids, cardiac glycosides and the crude plant drugs preparations to tackle their alkaloids are found in medicinal plants6. The health problems and it is perhaps due to their poor qualitative and quantitative variations of secondary economic conditions1. The number of investigators metabolites are not only varied between different determined nutritive value and mineral components of regions of the same plant but also vary in the different medicinal valued and emphasized the role of parts of the plants and also fresh and harvested nutrients, elements and heavy metals on the quality of products7-8. Medicinal plants were screened for herbal products and quality of food and growth bioactive compounds not only in India but also in products2-4. Similar studies were also carried out in other countries6,9,10. Dhyani et al3 attempted to study other countries. At least 25% of the perception drugs the edible plants at different valleys of Uttaranchal issued in the USA and Canada contained bioactive and emphasized the variation of proximates, compounds which are derived from plants5. In elemental components and nutritional value. They addition to primary metabolites, the secondary also discussed the importance of the plant resources in —————— the establishment of cottage industries to generate *Correspondent author: income and for the upliftment of social status of local E-mail: [email protected] Phone: : 9449704326; Fax: 91 082 82256262 inhabitancies. KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 399

Artocarpus gomezianus Wall. ex Trecul. var. on the average from the seashore. The average lakoocha Roxb., Monkey jack (Plate 1) is well known elevation ranges between 900 and 1500 m and some under-utilized fruits of Central Western Ghats. The places go beyond 2000 m. The region is rich in flora plant is an evergreen, tall and valuable timber yielding and fauna. The natural vegetation is interrupted by tree. The fruits are used in place of tamarind in the plantation of areca, coffee and paddy fields. The lower and higher middle altitudes of Central Western entire region receives heavy rain fall during monsoon Ghats. The entire plant is also studied with reference and the year is not clearly demarcated into different to food and fodder value in the neighbouring seasons. The fruits were collected from different countries of Bangladesh and Nepal11-14. The extensive regions between 20 and 2000 m altitudes. The studies have been carried out on screening, evaluation collected and identified plant material is deposited in and medicinal uses of fruits of this plant15-18. the department of Applied Botany, Kuvempu However, the phytochemistry and their variations University Shankaraghatta. Ashwatpura (Voucher with reference to altitudinal variation of Central specimen KUAB/AL-1, 22 altitudes, 12°52'N latitude, Western Ghats have not been carried out so far hence 74°53'E longitude) and Padubidare (Voucher an attempt is made to study the phytochemical specimen KUAB/AL-2, 147 altitude, 13.08°N properties of the fruits at different altitudes of Central latitude, 74.8°E longitude) come under coastal Western Ghats. regions, Banajalaya (Voucher specimen KUAB/AL-3, 579 altitude, 14.1667°N latitude, 75.0333°E Materials and Methods longitude) and Navanagere (Voucher specimen Collection of plant material and preparation of sample for KUAB/AL-4,590 altitude, 14.1667°N latitude, analysis 75.0333°E longitude) come under middle regions, Western Ghats extends about 1600 km starting in Gubburu (Voucher specimen KUAB/AL-5, North from Tapti river and going down to 763 altitude, 13.55°N latitude, 75.35°E longitude) and Kanyakumari in South. The Western Ghats, run Etinala (Voucher specimen KUAB/AL-6, parallel to the West coast of India about 40 km away 949 altitude, 12.97°N latitude, 75.78°E longitude)

Plate 1—a. Fruit bearing branch of Artocarpus gomezianus Wall. ex Trecul. var. lakoocha Roxb.; b., Mature ripe fruit; c. Halved unripe fruits; d. Halved ripe fruits 400 INDIAN J NAT PROD RESOUR, DECEMBER 2013

come under higher altitudes. The brief description of mixture was treated with 6 drops of Mayor’s the places along with average rainfall, temperature reagents/Wagner’s reagent/Dragondroff’s reagent. and altitude are given in Table 1.The average rainfall The formation of creamish precipitate/browinsh-red and temperature data are obtained from precipitate/orange precipitate indicated the presence meteorological department19.The coastal region is of respective alkaloids10, 20-22. thickly populated and plants are found in the cultivated and disturbed forests, whereas the plants in Phenol test The crude extract was mixed with 2 mL of 2 % the middle and higher altitudes are found in the semi solution of FeCl , a blue green or black coloration evergreen and the evergreen forests which are not 3 indicated the presence of phenols8, 21-22. disturbed or less disturbed. The mature unripe fruits samples were collected from above localities and the Ellagic acid test entire fruits were washed with water and dried in The crude extract was mixed with a few drops of shade. The dried fruits were grinded to powder and 5% mixture containing glacial acetic acid and 5% used for further analysis. sodium nitrate solution. A muddy yellow, olive brown, niger brown or deep chocolate colour I. Preliminary screening of secondary metabolites indicated the presence of phenols8, 21- 22. Extraction The shade dried fruit material was powdered using Flavonoids mixer grinder and subjected to Soxhlet extraction a. Shinoda test with petroleum ether, chloroform, 95% ethanol and 3 mL of each extract was treated with 5 mL of distilled water for 18 h in the order of increasing 95% ethanol and few drops of concentrated HCl and polarity of solvents.The condensed extracts 0.5 g magnesium turnings, formation of pink, were used for preliminary screening of reddish or brown colour indicated the presence of 8, 21-22 phytochemicals8. flavonoids .

Alkaloids b. Alkaline reagent test 2 mL of extract was treated with 1 mL of 1% HCl The crude extract was treated with 2 mL of 2 % and boiled for few minutes. 1 mL of the above solution of NaOH. An intense yellow colour formed

Table 1—Name of the places , altitude, average rainfall and average temperature in 2009 and 2010 S. No. Name of the Altitude Average rainfall in Average temperature Description of place place (MSL) (mm) in °C 2009 2010 2009 2010 1 Ashwatpura 22 3500 4200 27.5 26 The place lies in the coastal region of Karnataka. The plants are wild or grown in the private owned lands. 2 Padubidare 147 4180 3913 29 30.50 It also comes under the coastal region of Karnataka. The place is comparatively in the higher altitude. The trees are found not only in wild but also in the cultivated lands. 3 Banajalaya 579 1818.9 1827.6 27.5 26.5 The place also comes under the middle latitude of Central Western Ghats. The trees are tall and found in the forests of semi evergreen forest. 4 Navanagere 590 2886 2644 18.50 26 The place is also comes under the middle latitude of Central Western Ghats. The trees are tall and found in the forests of semi evergreen forest. 5 Gubburu 763 1905 1579 25 24 This place comes the under Central Western Ghats. The trees are found in the semi evergreen forests. 6 Etinala 949 989 858 30.50 26 It is comes under Hassan district of Karnataka and the place lies at the highest altitude of the present study. The tees are found in the coffee estates and it is said that the trees are very useful for shading of coffee plants. KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 401

which turned colourless on addition of few drops of Saponins diluted acid which indicated the presence of The crude extract was treated with 5 mL of flavonoids8, 21-22. distilled water in test tube. It was shaken vigorously. The formation of stable foam was taken as an 21-22 c. Zinc-hydrochloride acid reduction test indication for the presence of saponins . 4 mL of extract solution was treated with 1.5 mL of 50 % methanol solution. The solution was warmed Haemolysis test and metal magnesium was added. To this solution, 2 mL each of 18 % sodium chloride solution was 5-6 drops of concentrated hydrochloric acid was taken in two test tubes. 2 mL of distilled water was added. The appearance of red colour indicated the added to one test tube and the 2 mL of filtrate was presence of flavonoids and orange colour indicated added to another test tube. Few drops of blood was 8, 10, 20-22 added to the both the test tubes. Mixed and observed the presence of flavones . 21. 22 for haemolysis under microscope . d. Flavonoid test II. Quantitative analysis 5 mL of dilute ammonia solution was added to a portion of the aqueous filtrate of each plant extract A. Estimation of alkaloids A 500 mg of fruit sample was extracted with followed by addition of concentrated H SO .The 2 4 methanol and the methanol extract was condensed and appearance of yellow colour indicated the presence of 20 mL dilute acetic acid (1: 5) was added. The flavonoids. The yellow colouration disappeared on mixture was shaken well in a separating funnel. The standing. Few drops of 1% aluminium solution were acetic acid layer was collected and added with 25 mL added to portion of each extract. A yellow colouration N- hexane and chloroform. This was shaken again in indicated the presence of flavonoids8, 21-23. a separating funnel for 3 times and pH was adjusted to Tannins 8 using sodium hydroxide solution and shaken for Crude extract was mixed with 2 mL of 2% solution 30 min. In a separating funnel, the chloroform layer of FeCl3. A blue-green or black coloration indicated was collected, washed with water and pH was the presence of tannins8-10, 20-23. adjusted to 11 to 12 by adding ammonium hydroxide and the chloroform layer was collected and filtered Lignins test using dry filter paper. The filtrate was transferred to a When extract was treated with 2 % furfuraldehyde. pre-weighed beaker and dried under reduced pressure The formation of red colour indicated the presence of at 60ºC for 6h.The amount of alkaloids was calculated 8, 21, 22 lignins . using the following formula24.

Steroids Total alkaloid in percentage

Salkowski’s test The weight of alkaloidsresidue( x) The crude extract was mixed with 2 mL of = ×100 chloroform. Then 2 mL of concentrated H2SO4 was Weight of sample() W added carefully and shaken gently. The reddish brown 8, 21-22 x = Weight of the residue colour indicated the presence of steroids . y = Weight of the empty evaporator dish

Liebermann-Barchardt’s test z = Weight of the empty dish + alkaloid residue The extract was treated with a 2 mL acetic anhydride and mixed with 1 mL of concentrated Total (X) = Z – Y

H2SO4. Formation of blue-green ring indicated the presence of steroids20-23. B. Estimation of phenol by Folin Ciocalteu’s reagent method Phenols react with phosphomolybdic acid in Glycosides Folin-Ciocalteau reagent in alkaline medium and Crude extract was mixed with 2 mL of glacial produce blue coloured complex (molybdenum blue). acetic acid containing 1-2 drops of 2% solution of 500 mg of fruit sample was weighed and it was FeCl3.The mixture was poured into another test tube grinded with a pestle and mortar in 10 times volume containing 2 mL of concentrated H2SO4.The of 80 % ethanol. The homogenate was centrifuged at formation of the brown ring at the interphase 10.000 rpm for 20 minutes. The supernatant was indicated the presence of cardiac glycosides6,9,23. saved. The residue was re-extracted with five times 402 INDIAN J NAT PROD RESOUR, DECEMBER 2013

the volume of 80 % ethanol centrifuge and pooled the D. Estimation of tannin supernatants. The supernatant was evaporated to Tannin-like compounds reduce phosphotung- dryness. The residue was dissolved in a known stomolybdic acid in alkaline solution to produce a volume of distilled water (5 mL).The aliquots were highly coloured blue solution, the intensity of which pipette out (0.2 to 2 mL) in to test tubes. The final is proportional to the amount of tannins. The intensity volume was adjusted to 3 mL with distilled water is measured in a spectrophotometer at 700 nm. 0.5 g 0.5 mL of folin-Ciocalteu’s reagent was added. 2 mL of of powdered fruit material was weighed and 20 % Na2CO3 solution was added after 3 minutes. transferred to 250 mL conical flask. 75 mL of distilled Mixed thoroughly, the test tube was kept in water was added. The flask was boiled gently for boiling water for exactly one minute. Cooled and 30 minutes. Centrifuged at 2,000 rpm for 20 minutes the absorbance at 650 nm against a reagent blank. and supernatant was collected in 100 mL volumetric Simultaneously, a standard graph was prepared by flask and adjusted the final volume with distilled with different concentration of catechol. The phenol water. One mL of the sample extracted is transferred concentration was found out with standard graph to a 100 mL volumetric flask containing 75 mL water. and the phenol concentration was expressed as mg/100 g 5 mL of folin-Denis reagent and 10 mL of sodium fruit material. Finally the phenol is expressed carbonate solution were added and the final volume in percentage25. was adjusted to 100 mL with different water shaken well and the absorbance was read at 700 nm after C. Estimation of flavonoids 30 minutes (when absorbance was greater than 500 mg of fruit sample was added with 10 mL 0.7, make a 1+4 dilution of the sample). The blank methanol, homogenized and centrifuged at 3000 rpm was prepared with water instead of the sample. The for 10 minutes. The supernatant was used for the tannin content of the sample was calculated as tannic estimation of flavonoids. 1 mL supernatant was acid equivalents from standard graph. The tannin transferred to a 25 mL of conical flask and diluted to values are expressed in percentage27. 2 mL with distilled water, 4 mL of vanillin reagents E. Estimation of lignins was added rapidly from a burette (within 10-15 Refluxing the sample material with acid detergent seconds) to flask A. 4 mL of sulphuric acid was added solution removes the water-soluble and materials to flask B into flask C and 4 mL of vanillin reagent other than the fibrous component. The left-out and 2 mL of water was added and this was considered materials is weighed after filtration, dried, treated as blank. The content of flasks A and B were shaken with 72 % H SO and filtered, dried and ashed. The in a water bath below 30ºC and they were transferred 2 4 loss of weight on ignition gives the acid detergent to room temperature for exactly 15 minutes. The lignin. absorbance was measured at 500 nm against 47 % sulphuric acid (flask D). The absorbance of the a. Acid detergent fibre (ADF) contents of flask B and C were subtracted from that of 1 g of powdered fruit sample was placed in a round A. Alternatively; the absorbance of the content of bottom flask with 100 mL of acid detergent solution. A+D against B+C could be used. A standard curves Boiled the solution for 5 to 10 min. Heat was reduced was prepared using phloroglucinol and amount of to avoid foaming as boiling begins. Reflux for 1 h flavonoid (mg/g) was calculated. The flavonoids after the onset of boiling, boiling was carried out to values are expressed in percentage26. slow and even level. The container was removed swirled and filter the contents through a pre-weighed Calculation: Flavonoids (mg/g) = [(A-C) + (A-B) x sintered glass crucible (G-2) by suction and washed (A+B)-(B+C)] with hot water twice. The content was washed with Flask A- 1 mL of supernatant + 1 mL of water+4 mL acetone and broken up the lumps. The acetone of vanillin washing was continued until the filtrate was o colourless. The filtrate was dried at 100 C for Flask B- 1 mL supernatant+ 1 mL water+ 4 mL overnight. Cooled in desiccators and weighed. The sulphuric acid ADF content was expressed in percentage25.

Flask C- 2 mL water+ 4 mL vanillin (Blank) Weight of thefibre Percentageof ADF(%)= ×100 Flask D- 6 mL 47% H2SO4 Weight of thesample KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 403

b. Determination of acid detergent lignin (ADL) and basified with NH4OH (pH 11-12). It was The ADF content was transferred to a 100 mL extracted with chloroform (3x), condensed by beaker with 25-50 mL of 72% sulphuric acid. evaporation and used for chromatography. The 1 g asbestos was added allowed it to stand for 3 h alkaloid spots were separated using the solvent with intermittent stirring with a glass rod. The acid mixture chloroform and methanol (15:1). The colour was diluted with distilled water and filter with and hRf values of the separate alkaloids were pre-weighed Whatman No. 1 filter paper. The residue recorded both under ultraviolet (UV254nm) and visible and the glass rod were weighed several times to get light after spraying with Dragendroff’s reagent 8. rid of the acid. The filter paper was dried at 100ºC, cooled in desiccators and weighed. The filter was TLC study of phenol transferred to a pre-weighed silica crucible and ashed 1 g fruit sample was lixiviated in methanol on the filter paper with the content in a muffle furnace at rotatory shaker (180 thaws/min) for 24 h. The 550oc for about 3 h. The crucible was cooled in condensed filtrate was used for chromatography. The desiccators and weighed. The ash content was phenols were separated using chloroform and calculated. 1 g of asbestos was taken in blank 72% methanol (27:0.3) solvent mixture. The colour and H2SO4 was added and the steps were repeated as hRf values of these phenols were recorded under detailed above. The percentage of ADF was visible light after spraying the plates with 8 calculated with the help of following formula25. folin-ciocalteu’s reagent heating at 80ºC/10 min .

Weight of 72%H SO 2 4 Ash TLC study of flavonoids Washedfibre - ×100 1 g fruit sample was extracted with 10 mL (Test-Asbestosblank) (Test-Asbestosblank) methanol on water bath (60ºC/5 min). The filtrate was %ADL= Weightof sample condensed by evaporation, added a mixture of water and EtOAC (10:1 mL) and mixed thoroughly. The F. Estimation of sterol flavonoid spots were separated using chloroform and A 500 mL of sample was hydrolyzed by refluxing methanol (19:1) solvent mixture. The colour of hRf with 25 mL of 3 N HCl at 60ºC for 4 h; the solid values of these spots were recorded under ultraviolet 8 matter was filtered through Whatman filter paper No. (UV254nm) light . 1 and washed with dilute ammonia until the washing was neutral (pH 6.8-7.0) the sterol was extracted from TLC study of steroids the residue in Soxhlet extractor using chloroform. 2 g of powdered fruit samples was extracted with The 1 mL of chloroform extracts was evaporated to 10 mL methanol in water bath (80 ºC/15 min). The dryness and re-dissolved in 4 mL of sulphuric acid condensed filtrate was used for chromatography. The methanol reagent. After the reaction was initiated sterols were separated using chloroform, glacial acetic (2 min) the absorbance was noted at 405 nm against acid, methanol and water (64: 34: 12: 8) solvent blank (the optimum time required for a stable optical mixture. The colour of hRf values of these spots were density is 2 minutes). Standard graph was generated recorded under visible light after spraying the plates with anaisaldehyde sulphuric acid reagent and heating using the proper dilution of cholesterol and the 8 quantity of sterol (mg/g) presented was determined. (100ºC/6 min) . 27 The sterol concentration is expressed in percentage . TLC study of glycosides 1 g of fruit sample was extracted with 70% ethanol III. Separation of secondary metabolites by thin layer chromatography on rotary shaker (180 thaws/min) for 10 h. 70% lead For the thin layer chromatography studies of acetate is added to the filtrate and centrifuged at secondary metabolites pre-coated Alugram Sil 5000 rpm/10 min. The supernatant was further centrifuged by adding 6.3% Na CO at 10000 G/UV254 nm (Machery-Nagl GmbH, Germany) 2 3 aluminium plates (20x20 cm) were used. rpm/10 min. The retained supernatant was dried, dissolved in chloroform and used for chromatography. TLC study of alkaloids The glycosides were separated using EtOAC-MeOH- 1 g fruit sample was wetted with a half diluted H2O (80:10:10) solvent mixture. The colour and hRf NH4OH and lixiviate with EtOAC for 24 h at RT. The values of these spots were recorded by observation 8 organic phase was separated from the acidified filtrate under ultraviolet (UV254nm) light . 404 INDIAN J NAT PROD RESOUR, DECEMBER 2013

TLC study of saponins The successive extracts of A. gomezianus Wall. ex 1 g of fruit sample was extracted with 10 mL of Trecul. var. lakoocha Roxb. have revealed the 70% EtOH by refluxing for 10 min. The filtrate was presence of alkaloids, phenols, flavonoids, tannins, condensed, enriched with saturated n-BuOH and lignins, steroids, glycosides and saponins thus this thoroughly mixed. The butanol was retained, preliminary screening tests may be useful in the condensed and used for chromatography. The detection of the bioactive principles and subsequently saponins were separated using chloroform, glacial may lead to the drug discovery and development. acetic acid, methanol and water (64:34:12:8) solvent Further, these tests facilitate their qualitative mixture. The colour and hRf values of these spots estimation, quantitative separation, isolation, were recorded by exposing chromatogram to the purification and structural elucidation of secondary 8 iodine vapours . metabolites for pharmacologically active chemical compounds. Statistical Analysis The statistical analysis was made with the help of I. Preliminary phytochemical screening software28. The observations of various tests of different secondary metabolites are given below (Table 2). Results and Discussion The curative properties of medicinal plants are Alkaloids perhaps due to the presence of various secondary The petroleum ether, chloroform, ethanol and metabolites such as alkaloids, flavonoids, phenols, aqueous extract of fruit samples of different altitudes tannins, lignins, steroids, glycosides and saponins. are subjected for various tests (Mayor’s, Wagner’s

Table 2—Preliminary screening of secondary metabolites of extracts of fruits of monkey jack

Test for secondary 22 MSL 147 MSL 579 MSL 590 MSL 763 MSL 949 MSL metabolites PE CL ET AQ PE CL ET AQ PE CL ET AQ PE CL ET AQ PE CL ET AQ PE CL EA AQ ALKALOIDS 1. Wagner’s test - - + + - - + + - - + + - - + + - - + + - - + + 2. Dragenodorff’s test - - + + - - + + - - + + - - + + - - + + - - + + 3. Mayer’s test - - + + - - + + - - + + - - + + - - + + - - + + PHENOL 1. Phenol test - - + + - - + + - - + + - - + + - - + + - - + + 2. Ellagic acid test - - + + - - + + - - + + - - + + - - + + - - + + FLAVONOIDS 1. Shinoda’s test - - + + - - + + - - + + - - + + - - + + - - + + 2. Zinc-hydrochloride - - + + - - + + - - + + - - + + - - + + - - + + acid test 3. Alkaline reagent test + + - - + + - - + + - - + + - - + + - - + + - - 4. Flavonoid test + + - - + + - - + + - - + + - - + + - - + + - - TANNINS 1. Ferric chloride test - - + + - - + + - - + + - - + + - - + + - - + + LIGNINS 1. Lignins test - - + + - - + + - - + + - - + + - - + + - - + + STEROIDS 1. Salkowski test + + + + + + + + + + + + + + + + + + + + + + + + 2. Liebermann- - - + + - - + + - - + + - - + + - - + + - - + + Burchardt’s test GLYCOSIDES 1. Keller-Killiani test - + + + - + + + - + + + - + + + - + + + - + + + SAPONINS 1. Foam test - - + + - - + + - - + + - - + + - - + + - - - + 2. Haemolysis test - - + + - - + + - - + + - - + + - - + + - - - + PE–Petroleum ether: CL – Chloroform: ET – Ethanol: AQ – Aqueous KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 405

and Dragondroff’s) of alkaloids. Of the four extracts, Central Western Ghats. The tannins were screened by the extract of ethanol and aqueous showed +ve results number of investigators5,23,29. for all the fruit extract of different altitudes. Whereas Steroids the extract of petroleum ether and chloroform showed The steroids were screened by two tests namely –ve results. The number of investigators5,16,20 screened Salkowski’s test and Liebermann-Barchardt’s test. It is for alkaloids from the extracts of different solvents . interesting to note that all the extracts of solvents Phenols showed +ve results for steroids in test Salkowski’s test The plants have limitless ability to synthesize phenol whereas, the extracts of ethanol and aqueous showed and their derivatives. The presence of phenol in all +ve response and extracts of petroleum ether and 8 types of tissue is a characteristic of plants . The extract chloroform showed –ve results for Liebermann- of petroleum ether, chloroform, ethanol and aqueous Barchardt’s test. Vimalavady and Kadavul5 observed were subjected for phenol test by phenol test and -ve test for steroids from the extracts of petroleum ether, ellagic acid test. Similarly to that of alkaloids, the chloroform and ethanol, whereas, Velmurugan29 reported extracts of the ethanol and aqueous showed +ve results two types of steroids from the P. guajava and of the two to the test, whereas, petroleum ether and chloroform steroids, steroids I observed from only the hexane 5 showed the –ve results. Vimalavady and Kadavul extracts, whereas steroids II were observed from all the reported +ve results for the extracts of petroleum ether, extract of hexane, ethyl acetate and methanol. chloroform and also ethanol for the extract of leaves of Hugonia mystax. Velmurugan et al29 reported +ve test Glycosides for phenols for the root bark extract of hexane, ethyl The petroleum ether extracts showed –ve response, acetate, methanol of root bark of Psidium guajava L., whereas extracts of chloroform, ethanol and aqueous 23 showed +ve response to the fruit samples of all the Doss also reported +ve results for the phenols of 6 various medicinal plants of methanol extract. regions. Krishnaiah et al reported cardiac- glycosides from the six Malaysian medicinal plants. Flavonoids Saponins The petroleum ether, chloroform, ethanol and The saponins were screened with foam and aqueous extracts of fruit samples were screened for haemolysis test. The extract of petroleum ether and flavonoids by subjecting them to Shinoda test, chloroform showed –ve response, whereas extracts of alkaline reagent, zinc-hydrochloride acid reduction petroleum ether and aqueous showed +ve response not test and flavonoids tests. Of the four tests Shinoda test only for foam but also for haemolysis test. Krishnaiah showed +ve response to extracts of ethanol and 6 31 et al , Yadav and Munin Agarwala reported presence aqueous, whereas extract of petroleum ether and of saponins from various medicinal plants. chloroform showed +ve response for alkaline and flavones test. Various investigators6,7,30 screened II. Quantitative estimation of secondary metabolites flavonoids from various parts of the plants. Having screened for qualitative, secondary metabolites the quantitative estimation of alkaloids, Tannins phenols, flavonoids, tannins, steroids and lignins were The petroleum ether and chloroform extracts of made. The results revealed that there was a wide fruit samples showed –ve results, ethanol and aqueous variation of concentration of secondary metabolites at extracts showed +ve test at all the regions of the different altitudes (Table 3).

Table 3—Percentage of secondary metabolites at different altitudes /places of fruits of Artocarpus gomezianus var. lakoocha S. No. Factors/Places 22 MSL 147 MSL 579 MSL 590 MSL 763 MSL 949 MSL Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD 1 Alkaloids 11±1.00 37.00±3.00 7.7±0.38 26.67±3.40 15.33±2.21 10±2.00 2 Phenols 0.98±0.20 0.88±0.06 0.59±0.19 0.78±0.13 1.13±0.09 1.50±0.11 3 Flavonoids 0.41±0.21 0.66±0.33 0.73±0.50 0.53±0.17 1.28±0.33 0.46±0.16 4 Tannins 0.23±0.05 0.20±0.02 0.16±0.02 0.18±0.00 0.16±0.02 0.23±0.02 5 Steroids 0.23±0.05 0.20±0.02 0.16±0.02 0.18±0.00 0.16±0.02 0.25±0.03 6 Lignins 5.28±2.00 3.00±0.75 1.76±0.37 5.33±2.55 2.91±0.78 5.61±2.87 (Mean± SD: n=6) 406 INDIAN J NAT PROD RESOUR, DECEMBER 2013

Alkaloids alkaloids between 0.34 ± 0.1 and 1.04 ± 0.20 from the The percentage value of alkaloids varied between medicinal plants. Mallikarjuna et al8 observed the 7.7 ± 0.38 and 37.00 ± 3 at lower middle and higher variation of quantity of alkaloids in different parts of costal altitudes (Table 3). The study did not reveal the Strychnos potatorum L. and they recorded any ascending or descending pattern. Though, the percentage of alkaloids between 1.3 and 2.2. The percentage of alkaloids was higher at all the regions, quantity of alkaloids may vary not only in the parts the percentage of alkaloids varied remarkably among but also varied between fresh and preserved plant the different regions.. parts7,8. Of the six different sampling sites alkaloids are Alkaloids are nitrogen-containing compounds negatively correlated with rainfall and temperature at widely distributed in different plant group32. The Ashwatpura, Padubidare and Etinal region its alkaloids are the leading ligules of therapeutic significant at P≤ 0.05 level in both in 2009 and 2010 importance and they are heterocyclic indole sample. Whereas in case of positive correlation compounds which have proved to be having alkaloids are positively correlated with rain and pharmacological properties such as hypotensive, temperature at Banajalaya, Navanagere and Gubbur anticonvulsant, antiprotozoal, antimicrobial and region its significance is at P≤ 0.05 level in both 2009 antimalarial activities8. The biological properties of and 2010 sample (Table 4). alkaloids were studied33-37. The number of investigators estimated secondary metabolites in different parts of the medicinal plants6- Phenols 8,22. Doss23 recorded the percentage of alkaloids The percentage phenol ranged between 0.59 ± 0.19 between 0.28 ± 0.12 and 5.63 ± 0.20 in Berberis and 1.50 ± 0.11 at lower middle and higher altitudes, tinctoria Lesch. and Passiflora edulis Sims., respectively (Table 3). The percentage of phenols is respectively. Krishnaiah et al6 reported the percentage comparatively higher at higher and coastal regions of alkaloids between 0.24 ± 0.03 and 0.52 ± 0.12 in when compared to the middle altitudes. Similar to Emblica officinalis Gaertn. and Azadirachta indica, alkaloids, the percentage of phenols varied irregularly respectively. Edeoga et al9 reported the percentage among different altitudes.

Table 4—Simple correlation co-efficient between weather data and secondary metabolites of Artocarpus gomezianus var. lakoocha 2009 2010 Alkaloids Phenols Flavonoids Tannins Steroids Lignins Alkaloids Phenols Flavonoids Tannins Steroids Lignins Ashwatpura (22 MSL) Rainfall -0.998* -0.702 0.207 -0.968 -0.702 0.099 -1.000* -0.993 -1.000* 0.327 -0.982 0.945 Temperature 0.596 -0.217 -0.934 0.737 -0.217 0.842 -1.000* -0.993 -1.000* 0.327 -0.982 0.945 Padubidare (147) Rain fall 0.397 1.000* 0.981 0.564 -0.923 0.993 -0.134 -0.380 0.561 0.601 0.621 0.993 Temperature -0.721 0.350 0.953 -0.577 0.038 0.240 0.327 -0.756 0.874 0.176 0.908 0.240 Banajalaya (579 MSL) Rain fall 0.592 -0.975 0.554 0.504 0.504 0.391 0.102 0.645 0.811 -0.913 0.811 0.811 Temperature 0.500 0.189* 0.539 -0.993 -0.993 -1.000* 0.655 -0.999 -0.982 0.327 -0.982 -0.982 Navanagere (590MSL) Rain fall 0.118 -0.842 0.908 -0.900 0.072 -0.282 0.338 -0.123 0.675 -0.387 -0.444 -0.554 Temperature 0.500 -0.987 1.000* -0.655 -0.655 0.120 0.397 -0.060 0.721 -0.327 -0.500 -0.500 Gubburu (763 MSL) Rain fall 0.395 0.585 -0.984 0.225 0.650 0.481 1.000* -0.998* 1.000* 0.961 0.500 -0.606 Temperature 1.000* 0.508 -0.536 0.032 -0.593 -0.362 1.000* -0.687 -0.091 0.961 0.500 -0.581 Etinala (949 MSL) Rain fall 0.406 0.995 -0.997 -0.997 0.997 0.052 -0.444 0.444 -0.605 0.554 -0.605 0.239 Temperature -0.263 0.953* 0.955* 0.956* 0.955* 0.187 -0.993 0.993 -0.954 -0.596 -0.954 -0.833 KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 407

Of the six different sampling sites phenols Padubidare, Banajalaya and Navanagere in 2009 are negatively correlation with rainfall and sample and its significance is at P≤ 0.05 level. In case temperature at Ashwatpura, Banajalaya and of 2010 sample flavonoids are positively correlated Navanagere region and it is significant at P≤ 0.05 with rainfall and temperature at Padubidare, level in 2009 samples whereas in case of Banajalaya and Navanagere and it is significant at P≤ 2010 samples phenols are negatively correlated 0.05 level (Table 4). with rainfall and temperature at all the six different The percentage of flavonoids are studied by sites except Etinala region. In case of 2010 samples number of investigators8,9,23. The previous studies phenols are positively correlated with rainfall and reported flavonoids content below 1%. Mallikharjuna temperature at Padubidare, Gubburu and Etinala et al8 reported variations of flavonoids content in the region, it is significant at P≤ 0.05 in 2009 samples, different parts of the S. potatorum. whereas in case of 2010 sample phenols are positively The flavonoids are the group of naturally occurring correlated with rainfall and temperature at Etinala compounds, widely distributed as secondary region (Table 4). metabolites in the plant kingdom. They are known for 23 Doss recorded percentage of phenol between their antioxidant and antiradical properties32. 0.16 ± 0.10 and 12.85 ± 0.28 from different medicinal Flavonoids are hydroxylated, phenolic substance 15 plants. Krishnaiah et al reported variation of known to be synthesized by plants in reference to percentage of phenol between 0.024 ± 0.13 and microbial infection and they have been found to be 0.719 ± 0.23 in medicinal plants of Malaysia. Edeoga antimicrobial substance against wide array of 9 et al also observed variation of percentage of phenols microorganisms in vitro. The activity of flavonoids is 9 among medicinal plants. Mallikarjuna et al reported probably due to their ability to complex with variations of phenols from different parts of the extracellular and soluble proteins and complex with S. potatorum. bacterial cell wall. Phenols constitute a large class of compounds in which a hydroxyl group (-OH group) is bound to an Tannins aromatic ring. The phenolic compounds are one of the The concentration of tannin ranged between largest and most ubiquitous groups of plant 0.16±0.02 and 0.23±0.05 at lower middle and higher metabolites and they possess biological properties altitudes, respectively. At the same time the lower and such as ant atherosclerosis, cardiovascular protection higher altitudes recorded maximum percentage of and improvement of endothelial function, as well as tannins of 0.23±0.05 and 0.23±0.02, respectively. The inhibition of angiogenesis and cell proliferation quantitative variation of flavonoids at different activities. Natural antioxidant is due to the presence of sampling stations was very narrow. rich phenolic compounds such as flavonoids, phenolic Of the six different sampling sites tannins are 31 acid and tocopherol, etc . negatively correlated with rainfall and temperature at all the different places of Western Ghats except Flavonoids Gubbueu region. However, in case of 2010 sample The values of flavonoids ranged between tannins are negatively correlated with rainfall and 0.41±0.21 at lower coastal and lower higher altitudes, temperature at Banajalya Nvanagere and Etinala respectively (Table 3). The moderate values of region. In case of positive correlation tannins are flavonoids were recorded at lower and higher middle positively correlated with rainfall and temperature at latitudes. The low percentage of flavonoids is Gubburu region and it is significance at P≤ 0.05 level. recorded at lower and higher altitudes. Whereas in case of 2010 sample tannins are positively Of the six different sampling sites flavonoids are correlated with rainfall and temperature at negatively correlated with rainfall and temperature at Ashwatpura and Gubburu region and its significance Ashwatpura, Gubburu and Etinala region in 2009 is at P≤ 0.05 level (Table 4). The higher percentage of sample, whereas in case of 2010 sample flavonoids tannin was reported from the medicinal plants of are negatively correlated with rainfall and temperature Nigeria and Malaysia6,9. Tannins are water–soluble at Ashwatpura, Banajalaya, Gubburu and Etinala polyphenols and they have anti-carcinogenic, region. In case of positive correlation flavonoids are anti-mutagenic and antioxidative properties. They positively correlated with rainfall and temperature at protect cell from oxidative damage39. 408 INDIAN J NAT PROD RESOUR, DECEMBER 2013

Steroids with reference to altitudes, no specific ascending or Steroids ranged between 0.16±0.02 and 0.23±0.05 descending trend was observed. at lower middle and lower altitudes, respectively (Table 3). The quantitative variation of steroids was III. Separation of secondary metabolites by thin layer very narrow at different sampling stations. The range chromatography between minimum and maximum values of steroids The observation and hRf values of the various was very narrow. Of the six different sampling sites secondary metabolites are given in Table 6.

steroids are negatively correlated with rainfall and Alkaloids temperature at all the different places except Etinala Three quenching and fluorescing alkaloids spots region in 2009 sample. In case of 2010 sample were reported from the fruit extract of A. gomezianus steroids are negatively correlated with rainfall and var. lakoocha. Colour of the spots were green, temperature at Ashwatpura and Gubburu, whereas in blue and light green with respect to the hRf values case of positive correlation steroids are positively 38, 60 and 88, respectively (Table 6). Mallikharjuna correlated with rainfall and temperature at Etinala et al8 observed 20 quenching and fluorescing region in 2009 sample and it is significant at P≤ 0.05 alkaloids from the various part of the plant Strychnos level. In case of 2010 sample steroids are positively potatorum. The roots and stem bark recorded highest correlated with rainfall and temperature at Padubidare number of alkaloid spots when compared to seeds. and Gubburu region (Table 4). Again, it is interesting to note that the collected seeds recorded more number of spots when compared to the Lignins market seeds. Therefore, it may be attributed that the The values of lignins ranged between 1.76±0.37 quality and quantity of alkaloids not only varied with and 5.61±2.87 at lower middle and higher altitudes, respect to different individual plants but also among respectively (Table 3). The range between minimum the different parts of the same plant. Further, the and maximum values of lignins was very wide. The methods of processing and storage may be higher values of lignin were recorded at lower and important for quantitative and qualitative variation higher altitudes, respectively. Of the six different of alkaloids. sampling sites steroids are negatively correlated with rainfall and temperature at Banajalaya, Navanagere Table 5 a—The quantitative values of secondary metabolites on and Gubburu region in 2009 sample and it is the basis of variation with respective altitude significant at P≤ 0.05 level. In case of 2010 sample Place/Altitude Concentration of secondary lignins are negatively correlated with rainfall and metabolites temperature at Banajalaya, Navanagere, Gubburu and Ashwatpura/22MSL A>L>P>F>T≥S Etinala, whereas in case of positive correction lignins Padubidare/147 MSL A>L>P>F>T≥S are positively correlated with rainfall and temperature Banajalaya/579 MSL A>L>F>P>T≥S at Ashwatpurau, Padubidare and Etinala region in Navanagere/590 MSL A>L>P>F>T≥S ≥ 2009 sample. In case of 2010 sample lignins are Gubburu/763 MSL A>L>F>P>T S ≥ positively correlated with rainfall and temperature at Etinala/949 MSL A>L>P>F>S T Ashwatpura and Padubidare region (Table 4). Index: A= Alkaloids: L= Lignins: P= Phenols: F= Flavonoids: T= Tannins: S= Steroids The quantitative values of secondary metabolites are summarized in Table 5 a & b on the basis of Tables 5 b—Qualitative variation of individual secondary variation with respective altitude and qualitative metabolites Type of secondary Variation of secondary metabolites at variation of individual secondary metabolites. metabolites different place with their concentration The alkaloids were dominant secondary Alkaloids 147>590>763>22>949>579 metabolites followed by lignins, phenols, flavonoids, Phenols 949>763>22>147>590>579 tannins and steroids at lower, middle and higher Flavonoids 763>579>147>590>949>22 altitudes. There was a shifting between flavonoids and Tannins 22>949>147>590>579>763 phenols at higher middle and lower higher altitudes, Steroids 22>949>147>590>579>763 respectively. The amounts of tannin and steroids were Lignins 949>590>22>147>763>579 same at all the altitudes except at higher altitudes Index: 22 MSL= Ashwatpura: 147 MSL= Padubidare: 579 MSL= where steroid is higher than tannins. When secondary Banajalaya: 590 MSL= Navanagere: 763 MSL= Gubburu: metabolites are arranged on the basis of their quantity 949 MSL= Etinala. KRISHNAMURTHY & SARALA: PHYTOCHEMICAL STUDIES OF A. GOMEZIANUS VAR. LAKOOCHA 409

Table 6—Qualitative separation of alkaloids, phenols, flavonoids, 63 and 79 (Table 6). The qualitative separation of 8 glycosides, steroids and saponins by TLC (UV254nm) of glycosides of S. potatorum revealed 7 coloured spots Artocarpus gomezianus var. lakoocha with different hRf values. It is interesting to note that hRf Sl. the glycosides of root and stem bark were same and Colour spot Sample Solvent Rf (multiplication No. in collected seeds and market seeds were also same. with 100) Alkaloids The coloured spots which were found in root and stem 1 Green 2.3 6 2.3/6=0.38 38 (0.38×100) bark did not found in collected seeds and market 2 Blue 4 6 4/6=0.6 60 (0.6×100) seeds. At the same time coloured spots which were 3 Light green 5.3 6 5.3/6=0.88 88 (0.88×100) found in collected and marketed seeds were also not Phenols found in root and stem bark, respectively. 1 Blue 0.5 6.5 2.3/6.5=0.07 7 (0.07×100) 2 Dark Blue 1 6.5 4/6=0.6 10 (0.1×100) Steroids 3 Blue 1.5 6.5 5.3/6=0.88 20 (0.2×100) Flavonoids Presence of two sterols in the fruit samples was 1 Dark blue 3.5 6.5 3.5/6.5=0.53 53 (0.53×100) observed during present study. The colour of the spots 2 Yellow 4.5 6.5 4.5/6.5=0.69 69 (0.69×100) was light green and greenish black and hRf value of 8 3 Yellow blue 5.5 6.5 5.5/6.5=0.84 84 (0.84×100) and 66, respectively (Table 6). The qualitative Glycosides separation of sterols of S. potatorum8 revealed four 1 Light Green 2 6.3 2/6.3=0.31 31 (0.31×100) distinct spots with different colour and hRf values. 2 Pink 4 6.3 4/6.3=0.63 63 (0.63×100) 3 Pink 5 6.3 5/6.3=0.79 79 (0.79×100) The aerial part of the stem bark, seeds and market Steroids seeds contained maximum of four spots whereas the 1 Light Green 0.5 6 0.5/6=0.08 0.08×100) = 8 root sample contained three spots. However, the 2 Greenish 4 6 4/6=0.66 0.66×100 = 66 colour and hRf values were the same for the steroids black of root, stem bark, collected seeds and market seeds, Saponins 1 Intense respectively. 4.5 6 4.5/6=0.75 0.75×100 = 75 yellow Saponins Phenols One saponin with intense yellow colour and hRf Three phenol spots (blue, dark blue and blue) were values of 75 is observed (Table 6). The qualitative reported in the fruit samples and the hRf values is 7, S. potatorum8 10 and 20, respectively (Table 6). The investigation of separation of saponins of revealed that Mallikharjuna et al8 on qualitative separation of the coloured spots of saponins were same for different phenols revealed the interesting results. Of the plant parts with same coloured and hRf values. 11 coloured spots of phenols, the seeds contained The qualitative separation of secondary metabolites maximum of 9 and 7 spots from the market and by thin layer chromatography revealed three distinct collected seeds, respectively. In contract to alkaloids, coloured spots for phenols, flavonoids and glycosides phenols coloured spots were less in root and stem with different hRf values. The steroids have two and bark samples. The storage of seed may increase or saponins have one coloured spots with specific Rf derive new type of phenols. values. The above observations are useful for further isolation and purification of natural compounds. Flavonoids Three flavonoids spots were reported in the fruit Conclusion samples. Colour of the spots is dark blue, yellow and Monkey jack, an underutilized edible medicinal yellow blue and hRf values are 53, 69 and 84, and evergreen tree of Central Western Ghats is found respectively (Table 6). The thin layer chromatography between coastal and higher altitudes. The petroleum of flavonoids of S. potatorum revealed that the colour ether, chloroform, ethanol and aqueous extracts were spots appeared in the roots, collected seeds and screened for qualitative phytochemicals, preliminary market seed were same, whereas stem bark had metabolites and it is found that they were same. The separate flavonoids spots with different colours and quantitative estimation of secondary metabolites hRf values 8. revealed that alkaloids were dominant followed by Glycosides lignins, phenols, flavonoids, tannins, saponins and The three glycosides with light green, pink and steroids except at higher altitude where tannin was light pink spots were observed, having hRf values, 31, low. The qualitative separation of secondary 410 INDIAN J NAT PROD RESOUR, DECEMBER 2013

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