World Applied Sciences Journal 33 (12): 1908-1914, 2015 ISSN 1818-4952 © IDOSI Publications, 2015 DOI: 10.5829/idosi.wasj.2015.33.12.15625

Plant Physiology and Secondary Metabolites of Canistel ( campechiana)

1H. Mehraj, 2 R.K. Sikder, 34 U. Mayda, T. Taufique and 4 A.F.M. Jamal Uddin

1The United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8556, Japan 2Horticulture Development Division, BADC, Dhaka-1000, Bangladesh 3Department of Botany, Jahangirnagar University, Savar, Dhaka, Bangladesh 4Deaprtment of Horticulture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh

Abstract: Canistel () is a newly cultivated in Bangladesh. From this study, we tried to find out physiology and also secondary metabolites in hydro-alcoholic, methanol and aqueous extract of canistel fruit. Plant physiological characters were determined by respective instruments and fruit phytochemicals were screened using standard methods. SPAD reading (47.9%), photosynthetic rate (9.6 21 21 µmolm s ), stomatal conductance of H22 O (0.8 µmolm s ), CO references (348.4 vpm), H 2 O references as partial pressure (33.8 mBar) and P.A.R incident on leaf surface (271.1 µmolm 21s ) was found out. It yielded 123.6 kg fruit/plant while fruit had 11.6% degree of brix and 61.3 mg/100 g fruit Vit.-C with 13.3% moisture. Alkaloids, glycosides, , tannins, terpenoids, steroids, reducing sugar, phlobatannins, proteins, amino acids, lipids, and acidic compounds were found to be present but flavonoids, phenolics and saponins were found absent on canistel fruit. Canistel fruit have a number of secondary metabolites and identification of these phytochemicals may be helpful for phytochemists and pharmacologists in future.

Key words: Fruit Alcoholic Aqueous Extract Phytochemicals

INTRODUCTION MATERIALS AND METHODS

Canistel (Pouteria campechiana) is a new fruit for Experiment was conducted at Horticulture Bangladesh also known as yellow or egg-fruit Development Centre, Bangladesh Agricultural native to Central America but distributed to tropical and Development Corporation (BADC), Tangail and subtropical areas [1] that belongs to family. 2abiotech, Department of Horticulture, Sher-e-Bangla Dietary phytochemicals are found in different parts of the Agricultural University, Dhaka-1207, Bangladesh during like fruits, vegetables, legumes, grains, nuts, seeds, May 2012 to 2013. fungi, herbs and spices [2, 3]. Any fruits have the potentiality of excellent medicinal plant metabolites like Physiological, Physical and Chemical Properties of alkaloids, glycosides, terpenoids, tannins, phenolics, Plant: Leaf area and chlorophyll was measured by CL-202 flavonoids, steroids, saponins and lignans [4]. Canistel Leaf Area Meter and SPAD 502. Infrared gas analyzer fruit may also contain essential secondary metabolites. (IRGA)-LC pro+ Photosynthesis System was used to Generally, phytochemical surveys are done to identify the determine photosynthetic rate, stomatal conductance of chemical, toxicological and pharmacological aspects of a H22 O, CO references, H2 O references as partial pressure, plant or plant parts. Canistel plant physiology and fruit P.A.R incident on leaf surface. Brix percentages were secondary metabolites are till now unknown. So, we measured by Portable Refractometer (ERMA, Tokyo, conducted this study mainly to find out the plant Japan). Vit-C content was estimated by Oxidation physiology and fruit secondary metabolites of canistel Reduction Titration Method. 10 g sample was taken and fruits. dried in air oven at 105°C until to gain a constant weight

Corresponding Author: H. Mehraj, The United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8556, Japan. 1908 World Appl. Sci. J., 33 (12): 1908-1914, 2015 and dried sample weighted again. This procedure was yellow precipitate (with Hager’s reagent i.e., saturated repeated 5 times for accuracy. Moisture content (%) was picric acid) was regarded as positive for the presence of estimated by following formula: alkaloids.

Moisture (%) = {(W12 -W ) ÷ (W 10 -W )} × 100 Glycosides: By (a) FeCl3 , (b) Keller killiani’s and (c) Borntrager’s test (Anthraquinones)

Where, W01 = weight of empty petri dish (g); W = weight of petri dish with sample before drying (g) and W2 = (a) FeCl3 solution were added on extracts and dipped weight of petri dish with sample after drying (g). in boiling water for 5 minutes then cooled and shaken with equal volume of benzene. After that benzene Secondary Metabolites Determination: Canistel fruits layer was separated and ammonia solution was were washed with water and cut into small pieces, dried added. Rose-pink colour in ammoniacal layer and powdered. Extraction was performed by cold confirmed the presence of glycosides. (b) 2 ml of maceration method. glacial acetic acid with FeCl3 (1 drop) was added to 5

ml extract then underlayed with 1 ml of H24 SO (conc.). Hydro-alcoholic Extract and Aqueous Extract Brown ring denoted deoxysugar characteristic of Preparation: About 1000 g of fruit powder were immersed cardenolides and violet ring may appear below the in hydro-alcoholic (80% ethanol) solution in a 5000 ml flat brown ring. In acetic acid layer, greenish-blue ring bottom flask and cold extracted for 7 days with occasional may form throughout thin layer confirmed the shaking and warming. After that, clear filtrate was presence of cardiac glycosides [9]. (c) Benzene and obtained by Buchner funnel filtering. Filtrate was further 10% ammonia (10 ml of each) was added with 3 ml of concentrated by vacuum distillation, cooled, transferred extract. Pink, red or violet color in lower phase of into petri dish and dried in an oven at 60°C for five ammonia assured the presence of anthraquinone i.e., minutes. To remove excessive moisture hydro-alcoholic glycosides. extract was kept in a desiccator for 15 days [5]. Similarly aqueous extract was prepared only exception that fruit Carbohydrates: By (a) Molisch and (b) Fehling's test. powders were immersed on aqueous solution. (a) 0.5 mg of each extracts were dissolved in 5 ml Methanol Extraction: 10 g of fruit powder was dissolved distilled water and filtered individually. After treating in 100 ml of methanol and shake them on a rotary shaker the filtrates with 2 drops of alcoholic á-naphthol at 190-220 rpm for 24 h. Supernatant was collected slowly solution and H24 SO (2 ml conc.) violet ring was and stored at 4°C in airtight bottles. formed at junction which pointed out the presence of carbohydrates (Molisch’s Test). (b) Filtrates Qualitative Phytochemical Analysis: Phytochemicals hydrolyzing with diluted HCl, neutralizing with alkali were identified by testing hydro-alcoholic, methanol and and heating with Fehlings A and B solutions, aqueous extracts [6, 7, 8] as following the test included formation of red precipitation indicated the presence below. of carbohydrates (Fehling's test).

Alkaloids: By (a) Dragendroff's, (b) Wagner's, (c) Mayer's Flavonoids: By (a) Lead acetate (b) Alkali (c) Conc. H24 SO and (d) Hager's test (a-d) 0.5 g of extract was diluted to 10 treatment and (d) FeCl3. ml acid alcohol, boiled and filtered. Filtrate (5 ml) was added to dilute ammonia (2 ml) and chloroform (5 ml) then (a) and (b) Few drops lead acetate (for lead acetate shaken gently to extract alkaloidal base. Chloroform layer test) and NaOH (alkali test) solution was put in was extracted with 10 ml of acetic acid. This was divided extract solution then yellow color precipitate into four portions and Dragendroff's test, Wagner's test, formation directly indicated the presence of Mayer's test and Hager's reagents were added to each flavonoids (by lead acetate test) but if yellow color portion. The formation of cream (with Mayer’s reagent i.e., becomes colorless by the addition of dilute acid Potassium Mercuric iodide), brown/reddish brown (with confirmed the presence of flavonoids (by alkali test).

Wagner’s reagent i.e., Iodine potassium iodide and with (c) Conc. H24 SO (1 ml) and dilute ammonia (5 ml) was Draggendorff’s reagent i.e. potassium bismuth iodide) and added on the extract and appearances of the yellow

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color that disappear on standing was the indicator for Protein and Amino Acid: By (a) Xanthoproteic, (b) Biuret

presence of flavonoids. (d) Few drops of FeCl3 were and (c) Nin hydrin test added on extract then formation of blackish red color

indicating the presence of flavonoids. (a) Few drops of HNO3 (conc.) were added to the extracts. Yellow color observation indicated the

Tannins and Phenolics: By (a) FeCl3 and (b) gelatin test. presence of protein. (b) 1 ml NaOH (10%) were added

to the extract and heated. After that a drop of CuSO4 (a) Addition of 2-3 drops FeCl3 (2%) reagent to 1ml (0.7%) was added and formation of purplish violet of extract for formation of dark blue or greenish black colour confirmed proteins. (c) Distilled water (1 ml) color which indicated presence of tannins. (b) 2-3 and Nin hydrin (300 ìl) was added with extract (300 ìl) drops gelatin (10%) were added to 1ml extract and then boiled for 5-10 minutes. Dark purple color white precipitation assured the presence of phenolic indicated the presence of amino acids. compounds. Lipid and Test: A small quantity of powdered drug Terpinoids: By (a) terpine and (b) triterpine test. was rubbed on a clean and neat filter paper and observed for a permanent translucent strain. (a) Test for terpenoids was done by dissolving two or three granules of tin metal in 2 ml thionyl chloride Acidic Compounds: A bit of NaHCO3 was added with 300 solution and then, add 1 ml of the extract into the test ìl of extract. Gas bubbles ensured the presence of acidic tube. The formation of a pink colour indicates the compounds. presence of terpenoids. (b) Extracts were treated with Each test was done three times and positive results chloroform and filtered. The filtrates were treated with from each tests was marked by positive sign while a few drops of H SO (conc.), shaken and allowed to 24 negative result was marked by negative sign. stand. Golden yellow colour indicated the presence of triterpines. RESULTS AND DISCUSSION Steroids: By Salkowski test. About 5 mg extract was Canistel plant had 179.3 cm2 leaf area and 47.9% dissolved in 2 ml chloroform then 2 ml H SO (conc.) was 24 leaves SPAD reading while A (9.6 µmolm 21s ), g (0.8 added while red upper layer and yellow lower layer with s µmolm 21s ), C (348.4 vpm), e (33.8 mBar) Q (271.1 green fluorescence, indicated the presence of steroids. ref ref leaf µmolm 21s ) was also found (Table 1). Similar physiological characteristics were determined in Saponins: By foam test. About 1 ml of extract was taken which was diluted with 20 ml distilled water and then bougainvillea [10] and chilli [11]. Low chlorophyll content shaken in a graduated cylinder for 15 minutes. 1.0 cm layer in leaves caused higher yields also reduce heat load at top of foam indicates the presence of saponins [7]. canopy and may increase available nutrients for plant [12]. Stomatal conductance is important for CO2 gas exchanges Reducing Sugar: By (a) Fehling test and and outflow of water in vapor form because it also restricts the intake of CO2 [13]. More leaf photosynthesis (b) Molisch’s test. increases biomass productivity [14] and yield [15] but has (a) 0.50 g of fruit was added in 5 ml of distilled water co-relation on plant development phase [16, 17]. and 1 ml of ethanol was mixed. Fehling solution (1 ml Canistel fruit was 55.1 mm in length, 29.9 mm in of each Fehling solution A and B) was heated to boil diameter and 26.8 g of a single fruit weight whereas and poured it into aqueous ethanol extract. Color yielded 123.6 kg/plant. Fruits of canistel had 11.6% brix, reaction indicated the positive result. (b) Few drops 61.3 mg/100 g fruit Vit.-C. Moisture of the canistel fruit of Molisch’s reagent were added with dilute extracts was 13.3% (Table 1). and heated for 30 minutes. Brick red precipitate Canistel fruit’s were focused to qualitative confirmed the presence of reducing sugar. phytochemical screening for secondary metabolites like alkaloids, glycosides, carbohydrates, flavonoids, tannins Phlobatannins: Fruit powder solution was filtered 1% and phenolics, terpinoids, steroids, saponins, reducing aqueous HCl was added and then boiled by Hot plate sugar, phlobatannins, protein, acidic compounds, lipid stirrer. Red colored precipitate confirmed a positive result. and fat (Table 2). In the current study, hydro-alcoholic,

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Table 1: Some physiological, physical and chemical properties of canistel methanol and aqueous extracts showed similar results (Pouteria campechiana) fruits for canistel fruit. Secondary metabolites are Parameters characteristics Quantity Physiological properties of plant pharmacologically active [18] and also very important for Leaf area 179.3 cm2 disease prevention and health promotion. Similarly Chlorophyll content 47.9% phytochemical analysis was done in Asparagus spears Photosynthetic rate (A) 9.6 µmolm 21s 21 [19], citrus [20], Phaleria macrocarpa [21], medicinal Stomatal conductance of H2 O (gs) 0.8 µmolm s

CO2 references (Cref) 348.4 vpm plants [22], Acalypha ornate [23], Albizzia lebbeck [24], H2 O references as partial pressure (eref ) 33.8 mBar Ocimum americanum [25], Nerium oleander and P.A.R incident on leaf surface (Q ) 271.1 µmolm 21s leaf Momordica charantia [26], Pyrus pashim [27], Jatropha Physical and chemical properties of fruit Fruit length 55.1 mm [28] and various plant parts of Quetta Balochistan [29, Fruit diameter 29.9 mm 30]. They all mentioned the presence/absence of alkaloids, Single fruit weight 26.8 g glycosides, carbohydrates, flavonoids, tannins, terpenes, Yield/plant 123.6 kg Brix 11.6% phenolics, steroids, saponins, reducing sugar, Moisture 13.3% phlobatannins, protein, amino acid, lipid and fat also Vit-C 61.3 mg/100 g fruit amino acid. Alkaloid has pharmacological activities [31]

Table 2: Presence or absence of secondary metabolites of canistel fruits because it poses anti-bacterial and analgesic properties Test results for hydro-alcoholic, [32] also used for creation of potent pain killer [33]. Name of the tests methanol and aqueous extract Remarks Glycosides are cardio active drugs [34] because it inhibits Alkaloid Na++ and K pump by rising availability of Na+ and Ca + to Mayer’s reagent ++ Alkaloid present Wagner’s reagent ++ heart muscles which gets better cardiac output and lessen Dragendroff’s reagent ++ heart expansion [35]. Presence of alkaloids and glycoside Hager's test ++ on canistel fruit (Table 2) is very important and dietary Glycoside intake may prevent heart failure and cardiac arrhythmia. FeCl3 test ++ Glycoside present Keller killianis test ++ Flavanoids and tannins are used for the treatment of Borntrager’s test -- intestinal disorders also have anti-microbial, anti- inflammatory, anti-allergic, anti-cancer, anti-neoplastic Molisch’s test ++ Carbohydrate present Felhing’s test ++ activities [36]. Tannins also act as primary antioxidants or Flavonoid free radical scavengers that may make the canistel Lead acetate test -- Flavonoid absent antioxidant capacity and is able to inhibit HIV [37]. Dietary Alkali test ++ flavonoids also inhibit heart diseases, inflammation and Conc. H24 SO test --

FeCl3 test -- tumor growth but phytochemical analysis of the canistel Tannins and phenolics fruit did not show the positive result for flavonoids while FeCl3 test ++ Tannic present but tannins were found to be present on canistel fruit (Table phenolics absent Gelatin test -- 2). Terpinoids are natural lipids and many of them are Terpenoid commercially important due to their flavors and Test for Terpine ++ Terpenoid present fragrances; also important for agricultural produces that Test for Triterpine ++ ensure flavor [38]. It acted as a phytoalexins in plant Steroid alkowski’s test ++ Steroid present defense against natural enemies [39] also have medicinal Saponin properties like anti-carcinogenic, anti-ulcer, hepaticidal, Foam Test -- Saponin absent anti-microbial or diuretic, anti-malarial drug and anti- Reducing sugar cancer drug [40] while terpenes may act as Fehling test ++ Reducing sugar present Molisch’s test ++ phytohormones of plants also slower the cancer cell Phlobatannins ++ Phlobatannins present growth. Presence of terpenoid in canistel fruit (Table 2) Protein and amino acid indicated that consumption of this fruit may protect the Xanthoproteic test ++ Protein present Biuret test ++ Bangladeshi people from some risky diseases. Dietary Nin hydrin test ++ Amino acid present intakes of steroid can increases anti-microbial [36], anti- Lipid and Fat ++ Lipid and fat present bacterial [41, 42] and anti-viral [43] properties in human Acidic compounds ++ Acidic compounds present body and that were present in canistel fruit (Table 2). ++ = present and -- = absent Saponins played important role for hypercholesterolemia,

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