Isolation and characterization of secondary metabolites from leaves of

Table of contents List of Abbreviation ............................................................................................................................ i List of Figures ..................................................................................................................................... ii List of schemes ................................................................................................................................... iii 1. INTRODUCTION .......................................................................................................................... 1 2. LITERATURE REVIEW ................................................................................................................ 2 2.1.

Taxonomical classification ....................................................................................................... 2

2.2.

Vernacular names ...................................................................................................................... 2

2.3.

Plant description........................................................................................................................ 2

2.4.

Geographical distribution .......................................................................................................... 3

2.5.

Phytochemical constituents of C. papaya L.............................................................................. 4

2.6.

Pharmacological properties of Carica papaya L. ..................................................................... 9

3. RATIONALE ................................................................................................................................ 12 4. AIM AND OBJECTIVES ............................................................................................................ 13 4.1.

Aim ......................................................................................................................................... 13

4.2.

Objectives ............................................................................................................................... 13

5. MATERIALS AND METHODS. ................................................................................................ 14 5.1.

Plant material .......................................................................................................................... 14

5.2.

Chromatographic materials ..................................................................................................... 14

5.3.

Instruments .............................................................................................................................. 14

5.4.

Derivatizing reagents .............................................................................................................. 14

5.5.

Extraction method ................................................................................................................... 15

5.6.

TLC profiling of aqueous methanolic extract ......................................................................... 17

5.7.

VLC fractionation of alkaloids rich extract ............................................................................ 18

5.8.

TLC profiling of VLC fractions ............................................................................................. 19

5.9.

Column chromatography......................................................................................................... 19

6. RESULTS AND DISCUSSION ................................................................................................... 23 7. SUMMARY ................................................................................................................................... 28 8. REFERENCES ............................................................................................................................. 29

List of abbreviation cm

Centimeter

λ

Lambda

β

Beta

µ

Micron

δ

Delta

d

Doublet

dd

Doublet of Doublet

HPLC

High Performance Liquid Chromatography

DEPT

Distortionless Enhancement by Polarization Transfer

2D NMR

Two Dimensional Nuclear Magnetic Resonance

PDA

Photo Diode Array

Rf

Retention Factor

Hz

Hertz

LOD

Limit of Detection

LOQ

Limit of Quantitation

IR

Infra-red Spectroscopy

J

Coupling constant

S

Singlet

T

Triplet

m/z

Mass to charge ratio

µg/ml

Microgram per Mililiter

µM

Micromolar

TMS

Tetramethylsilane

TLC

Thin Layer Chromatography

i

List of Figures Figure 1: C. papaya Linn: (A) Papaya tree; (B) Papaya Leaf. ...................................................................... 1 Figure 2: Enzyme present in leaves of Carica papaya L. ............................................................................ 6 Figure 3: Alkaloids present in leaves of Carica papaya L. .......................................................................... 6 Figure 4: Flavonoids and flavonoid glycosides present in leaves of Carica papaya L. ............................... 7 Figure 5: Phenols and phenolic acid present in Carica papaya L. ............................................................... 8 Figure 6: TLC profile of methanolic extract ............................................................................................... 17 Figure 7: Comparative TLC profiling of ethanolic and methanolic extract of Carica papaya L. .............. 17 Figure 8: VLC fractions of MeOH alkaloids rich extract. .......................................................................... 18 Figure9: TLC Profile of VLC fractionation. ............................................................................................... 19 Figure 10: TLC profiling of isolated compound ......................................................................................... 21 Figure 11: Comparative TLC profiling of β-sitosterol as standard and isolated compound ..................... 23 Figure 12: 1H NMR spectrum of compound (IPGJ-12) .............................................................................. 24 Figure 13:

13

C NMR spectrum of compound (IPGJ-12) ............................................................................ 25

Figure 14: DEPT-135 carbon NMR spectrum of compound (IPGJ-12) .................................................... 25 Figure 15: Direct mass spectrum of isolated compound (IPGJ-12) ........................................................... 26 Figure 16: HPLC chromatogram of isolated compound (IPGJ-12) ............................................................ 27

ii

List of schemes Scheme 1: A schematic representation of extraction procedure. ................................................................ 16 Scheme 2: A schematic representation of column chromatography. .......................................................... 20 Scheme 3: A schematic representation of column chromatography. .......................................................... 22

iii

Isolation and characterization of secondary metabolites from leaves of Carica papaya Linn.

1. INTRODUCTION Papaya (Carica papaya Linn) is known as powerhouse of nutrients and is available throughout the year. During the last few years, major insight has been achieved regarding the biological activity and medicinal application of papaya and now it is considered as valuable nutraceutical fruit plant because of its high contents of nutrients and medicinal values as well. It is a rich source of three powerful antioxidants vitamin C, vitamin A and vitamin E, minerals like magnesium and potassium, vitamin B, pantothenic acid, foliate and fiber. In addition to all this, it contains a digestive enzyme - papain that effectively treats causes of trauma, allergies and sports injuries. All the nutrients of papaya as a whole improve cardiovascular system, protect against heart diseases, heart attacks, strokes and prevent colon cancer. The fruit is an excellent source of beta-carotene that prevents damage caused by free radicals that may cause some forms of cancer. It has been reported that it helps in the prevention of diabetic heart disease. Papaya lowers high cholesterol levels as it is a good source of fiber. Carica papaya (C. papaya) L. is one of the major fruit crops cultivated in tropical and sub tropical zones. Worldwide over 6.8 million tons of fruit were produced in 2004, of this volume, 47% was produced in Central and South America (mainly in Brazil), 30% in Asia and 20% in Africa. The papaya industry in Brazil is one of the world’s largest that continues to show rapid growth. Although papaya is mainly grown (>90%) and consumed in developing countries, it is becoming an important fruit internationally and is consumed both as a fresh fruit and as processed products.

Figure 1: C. papaya Linn: (A) Papaya tree; (B) Papaya Leaf.

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2. LITERATURE REVIEW Taxonomical classification,1 vernacular name3 and plant description4, 5 of papaya are as follows: 2.1. Taxonomical classification Kingdom

Plantae

Division

Magnoliophyta

Class

Magnoliopsida

Order

Brassicales

Family

Caricaceae

Genus

Carica

Species 2.2. Vernacular names

Carica papaya L.

English

Papaya, papaw

Hindi

Papita

Sanskrit

Arand-kharpuja

Bengali

Papeya, papayabaum

French

Papaya

Marathi

Pappay

2.3. Plant description 2.3.1. Habitat and leaf description

2

Height

10 to 15 feet

Spread

5 to 7 feet

Plant habit

Upright

Growth rate

Fast

Texture

Coarse

Leaf arrangement

Alternate

Leaf shape, color

Star-shaped, green

Leaf venation

Palmate

Leaf blade length

18 to 36 inch


2.3.2. Fruit

2.3.3.

Fruit shape

Oval

Fruit length

6 to 12 inch

Fruit cover

Fleshy

Fruit color

Orange yellow

Fruit characteristic

Suited for human consumption

Trunk and Branches

Trunk/bark/branches Usually with one stem/trunk

2.3.4.

Twig color

Green

Twig thickness

Thick culture

Light requirement

Plant grows in full sun

Soil tolerances Soil pH

Slightly alkaline

Sand; loam

Clay

Soil salt tolerances

Poor

Plant spacing

36 to 60 inches

2.4. Geographical distribution Although the exact area of origin is unknown, papaya is believed to be native to tropical America, perhaps Southern Mexico and neighboring Central America. Successful commercial production is done primarily in Hawaii, Tropical Africa, the Philippines, India, Ceylon, Malaysia and Australia, apart from the widespread but smaller scale production in South Africa, and Latin America. In India, papaya is cultivated in Maharashtra, Bengal, Bihar, Haryana, Punjab, Delhi, Andhra Pradesh and Uttar Pradesh.6, 9 The varieties of papaya have been described blow. a) Mexican Red Reddish to Red – Orange in color, more elongated in shape and have red flesh.

3


b) Solo Most common variety, this variety doesn’t produce any male tree, Reddish - Orange flesh, pear shaped. c) Sunrise Solo Pear shaped fruit, smooth skin, Reddish Orange color, sweet, sugar Content high. d) Sunset Solo Small to medium sized, pear shaped, Orange Red etc.1, 2 2.5. Phytochemical constituents of C. papaya L. The phytochemical study of Carica papaya Linn. revealed the presence of different type of the chemical compounds like alkaloids, flavonoids, quinones, saponin glycosides, enzymes, amino acids, phenols and phenolic acid, organic acids, and others like creatine, choline, trigonelline etc. Table 1: Phytochemical constituents in various part of Carica papaya L. Part

Constituents in Carica papaya Linn.

Fruit

Thiamine, riboflavin, lycopene, and carotene, amino acids, citric and malic acids (green fruits), volatile compounds: linalool, benzylisothiocyanate, cis and trans 2, 6-dimethyl-3,6 epoxy-7 octen-2-ol, Alkaloid, α; carpaine, benzyl- β -D glucoside, 2-phenylethyl -β-D-glucoside, 4-hydroxy- phenyl-2 ethyl-β-Dglucoside and four isomeric malonated benzyl-β-D-glucosides17

Bark

β -Sitosterol, carpaine, glucose, fructose, sucrose, galactose and xylitol2

Seed

Carpaine, benzylisothiocyanate, benzylglucosinolate, glucotropacolin, benzylthiourea, hentriacontane, β-sitosterol, caricin and myrosin7

Root

Carposide and an enzyme myrosin4

Leaf

Alkaloids, (carpain, pseudocarpain and dehydrocarpaine) I and II, flavonoids, phenols and phenolic acid, choline, carposide, vitamin C and E5, 23

Latex

Proteolytic enzymes, papain and chemopapain, glutamine cyclotransferase, chymopapains A, B and C, peptidase A and B13

4


Where some compounds such as asprotocatechuic acid, p-coumaric acid, caffeic acid, chlorogenic acid, kaempferol and quercetin were detected and identified in qualitative analysis, the quantitative analysis showed the presence of phenolic acids as the main compound, while chlorogenic acid was found in trace amounts, compared to the flavonoids and coumarin compounds. The quantities detected were 0.25 mg/g (dry leaf) for caffeic acid, 0.33 mg/g for p-coumaric acid and 0.11 mg/g for protocatechuic acid. Kaempferol and quercetin were 0.03 and 0.04 mg/g, respectively, while 5,7-dimethoxycoumarin was 0.14 mg/g. 4-9, 15 2.5.1. Enzymes present in Carica papaya L. Latex tapped from green papaya fruit (Carica papaya L.) is a rich source of the enzyme papain, chymopapain, caricain, several of these enzymes belong to class-II and a class-III chitinase, and related cysteine endopeptidases (CPs). Crude latex and industrial papain preparations also contain other proteinase-stable enzyme activity such as acid phosphatase, amylase,

chitinase,

endo-1,3-β-glucanase,

glutamine

cyclotransferase,

lysozyme,

peroxidase, and lipase. Non-CPs enzymes purified and characterized from papaya latex include a chitinase glutamine cyclotransferase. Purification of these papaya enzymes calls on the use of ion-exchange supports (such as SP-Sepharose Fast Flow) and hydrophobic supports. The use of covalent or affinity gel is recommended to provide preparations of cysteine endopeptidase with a high free thiol content (ideally 1 mol of essential free thiol function per mol of enzyme). The selective grafting of activated methoxypoly (ethylene glycol) chains (with mol wt. of 5000) on the free thiol function of the proteinases provides an interesting alternative to the use of covalent and affinity chromatography especially in the case of enzymes such as chymopapain that contains in its native state, two thiol functions.12,13,14 Papain i.e. a protein-digesting enzyme obtained from unripe papaya fruit is used to tenderize meat and as a food supplement to aid digestion. Lycopene is a red carotenoid pigment present in Carica papaya L. Two major new piperideine alkaloids, dehydrocarpaine I and II have been reported in the leaves of Carica papaya L. and have higher concentration than carpaine. Various phytochemical constituents present in Carica papaya L. are shown below.

5


Figure 2: Enzyme present in leaves of Carica papaya L.

Carica papaya leaves contain carpaine as one of the major alkaloid components along with two new piperidine alkaloids, dehydrocarpaine I and II. The alkaloid carpaine is a dimeric, macrocyclic lactone with two piperidine units incorporated into the ring. In addition to carpaine(C28H50N2O4) which consists of a 28-membered ring, this plant also has azimine and azicarpaine, the corresponding 22- and 24-membered ring analogues of carpaine.23 Emetine is also one of the alkaloid present in leaves of Carica papaya L.5, 23

Figure 3: Alkaloids present in leaves of Carica papaya L.

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Carica papaya L. leaves also contain flavonoids and flavonoid glycoside. Flavonoid glycosides are a common component of many plants and are a significant class of archetypal plant constituents with various pharmacological activities as a digestive, diuretic, antioxidant, laxative, hypotensive, and for the treatment of hepatic and dermatological complications.

Figure 4: Flavonoids and flavonoid glycosides present in leaves of Carica papaya L.

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The leaves of Carica papaya L. have a major quantity of phenols and phenolic acids. Natural phenolic compounds play an important role in cancer prevention and treatment. Phenolic compounds from medicinal herbs and dietary plants include phenolic acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, and others. Various bioactivities of phenolic compounds are responsible for their chemopreventive properties (e.g, antioxidant, anticarcinogenic, or antimutagenic and anti-inflammatory effects. The quantity of some reported phenols and phenolic acids in dry leaves of Carica papaya L. are chlorogenic acid (0.11mg/g), ferulic acid (0.19mg/g), gallic acid (0.35mg/g), 5,7 dimethoxycoumarin (0.33mg/g), caffeic acid (0.25mg/g), o-coumaric acid (0.29mg/g), p-coumaric acid (0.33mg/g), protocatechuic acid (0.11mg/g) respectively.13, 14, 19, 24

Figure 5: Phenols and phenolic acid present in Carica papaya L.

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2.6. Pharmacological properties of Carica papaya L. The plant Carica papaya has been proved for various medicinal activities like antioxidant, anti-hypertensive, wound healing, hepato-protective, anti-inflammatory, antimicrobial, antifungal, anti-fertility, anti-histaminergic, diuretic, anti-amoebic, anti-tumor, anthelmintic, effect on smooth muscles, antimalarial, hypoglycemic activity, immunomodulatory activity, anti-ulcer activity, anti-sickling activity.2,4,10,17,18,20,21 2.6.1. Antioxidant activity The methanolic extract of unripe fruits of Carica papaya was evaluated in vivo for its effect on activities of some antioxidant enzymes which includes glutathione peroxidase (GPx), glutathione transferase (GST), glutathione reductase, catalase and glucose-6-phosphate dehydrogenase in mice given an oral dose of 100 mg/kg. There was significant increase in the activities of glutathione reductase, GST, GPx, glucose-6-phosphate dehydrogenase due to the ethyl acetate fraction. Significant decrease in GPx was observed in kidney following administration of ethyl acetate fraction. It was suggested that quercetin and β-sitosterol may be responsible for the antioxidant potential.10,13 2.6.2. Anti-hypertensive activity The methanolic extract (20mg/kg i.v) of ripe fruit of C. papaya was used for the evaluation of anti-hypertensive activity in comparison of enalapril. The antihypertensive effects elicited by the methanolic extract of C. papaya were similar to those of enalapril, and the sensitivity was normalized in treated spontaneously hypertensive rats.26 2.6.3. Wound healing activity The aqueous extract of C. papaya fruit [100 mg/kg for 10 d] was tested for wound healing property in streptozotocin induced diabetic rats using excision and dead space wound models. The aqueous extract showed 77% reduction in the wound area when compared to 59% contraction to wound of the controls.10

9


2.6.4. Hepatoprotective activity The aqueous (250 mg/kg, p.o) and ethanol (250 mg/kg, p.o) extracts of C. papaya showed significant hepatoprotection by lowering the biochemical parameters such as SGPT, SGOT, serum bilirubin, akaline phosphatase.2, 5 2.6.5. Anti-inflammatory activity The anti-inflammatory activity of an ethanolic extract of Carica papaya leaves was investigated in rats using carrageenan induced paw oedema, cotton pellet granuloma and formaldehyde induced arthritis models. Experimental animals received 25–200 mg/Kg (orally) of the extracts or saline (control group) and the reference group received 5 mg/Kg of indomethacin. reduced paw oedema in the carrageenan test. Likewise the extract produced significant reduction in the amount of granuloma formed from 0.58 ±0.07 to 0.22 ±0.03 g. In the formaldehyde arthritis model, the extracts significantly reduced the persistent oedema from the 4-10 day of the investigation.25 2.6.6. Antimicrobial activity The aqueous extract of C. papaya leaves and roots at different concentrations (25, 50, 100, 200 mg/mL) showed antimicrobial activity against some human pathogenic bacteria using the agar diffusion method.14 2.6.7. Antifungal activity The latex of C. papaya and fluconazole has synergistic action on the inhibition of Candida albicans growth. This synergistic effect results in partial cell wall degradation. Latex proteins appear to be responsible for antifungal action and minimum protein concentration for producing a complete inhibition was reported as about 138 mg/mL.7, 9 2.6.8. Anti-fertility activity The influence of the crude aqueous extract of Carica papaya L. (Caricaceae) seeds has been studied on semen profile, fertility, body and organ weight response, and toxicology in male albino rats. The extract was administered at the dose regimens of 10 and 50 mg/animal/day orally for 30, 60, and 90 days and 0.1 and 1.0 mg/animal/day intramuscularly for 15 and 30 days. Cauda epididymal sperm motility and count was reduced significantly at low and high dose regimens both in the oral as well as the intramuscular groups.6

10


2.6.9. Diuretic activity Aqueous root extract of C. papaya when given orally at the dose of 10 mg/kg to rats produced significant increase in urine output and showed similar profiles of urinary electrolyte excretion to that of hydrochlorothiazide.21 2.6.10. Anti-malarial activity The petroleum ether extract of the rind of raw papaya fruit at concentration ranging from 0.05-1000 µg/ml exhibited significant anti-malarial activity.1, 5 2.6.11. Hypoglycemic activity The ethanolic leaf extract of Carica papaya at the dose 5.0 mg/ kg produced significant blood sugar level reduction with no significant effects at higher dose of 10 mg/kg. The extract delayed the onset of hypoglycemic activity of glimepiride and increased the hypoglycemic effect of metformin with the variables interacting differently for each drug extract combinations.18,21 2.6.12. Ameliorative effect of leaves of C. papaya in hepatotoxicity The study was performed by administering the aqueous extract of Carica papaya leaves at the dose of 400 mg/kg in rats prior to administration of ethanol or combination of isoniazid and rifampicin. The effects on the levels of serum indicators of liver damage (ALT, AST, alkaline phosphatase and total bilirubin) and tissue antioxidant parameters like TBARS, GSH and SOD were determined. Histopathology of liver was also performed to study the influence of drug on tissue integrity.1 2.6.13. Anti-ulcer activity Aqueous seed extract of Carica papaya was administered to rat at the dose of 50 mg/ kg and 100 mg/kg p.o and its effect on alcoholic induced acute gastric damage and blood oxidative stress was determined. The rat treated with 100 mg/kg of extract showed significant reduction in gastric acidity.17

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3. RATIONALE Papaya (Carica papaya L.) is a popular and important fruit tree in tropical and subtropical parts of the world. The fruit is consumed worldwide as fresh fruit and vegetable or used as processed product. The many benefits of papaya can be attributed to its high content of vitamin A, B and C, proteolytic enzymes like papain and chymopapain which have antiviral, antifungal and antibacterial properties, and many secondary metabolites like alkaloids, flavonoids, flavonoid glycosides, phenolic and polyphenolic compounds which have various pharmacological activities. During the last few years, major insight has been achieved regarding the biological activity and medicinal application of papaya and now it is considered a valuable nutraceutical fruit plant.

12


4. AIM AND OBJECTIVES 4.1. Aim Isolation and characterization of secondary metabolites from leaves of Carica papaya Linn. 4.2. Objectives 1. Collection & authentication of plant material 2. Preparation of aqueous methanolic extract 3. TLC profiling of prepared extract 4. Isolation and identification of compounds from aqueous methanolic extract of Carica papaya L. leaves using various chromatographic techniques 5. Characterization of isolated compounds using modern spectroscopic methods

13


5. MATERIALS AND METHODS. 5.1. Plant material Carica papaya L. leaves were collected from residential area of National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab-160062 with the specimen No. NIP-H-263. The leaves were dried in shade for 15 days. The total weight of powder of dried leaves was 1.1kg. 5.2. Chromatographic materials Pre-coated TLC plates having silica gel 60 F254 thickness 0.25 mm (Merck Co. ltd.), Silicagel 60-120#, 230-400# were used for column chromatography. Silica gel G and Silica gel GF254 was used for preparative TLC. All the solvents used for extraction were of laboratory grade are (Methanol (MeOH), water, ethyl acetate (EtOAc), hexane, chloroform (CHCl3), acetic acid. 5.3. Instruments Plant material was extracted by using Soxhlet extractor (Perfit India Ltd.). Extract was concentrated using vacuum rotary evaporator (Buchi R-114, Switzerland). Mass spectras were on a LCMS (Water U.S.A). 1H NMR spectra were recorded on 400 MHz spectrometer and 13C NMR spectra were recorded on 100 MHz (Brukers Ultra shield TM) with TMS as an internal standard. The HPLC analysis was carried out on a Kromasil (Phenomenex) C18 column (250 × 4.6mm) connected to a Shimadzu HPLC system (LC-10AT VP) fitted with SIL-20AC Autosampler and SPD-M10A VP photodiode array detector. Class VP software (Shimadzu) was used both for data collection and integration. The Melting point was taken by digital melting point apparatus (Perfit india Ltd.). Sonicator was used to sonicating the extract (Bandelin sonorex digitec). 5.4. Derivatizing reagents a. Dragendorff's reagent- Pour 0.5 g of bismuth nitrate into an empty beaker and add 10

ml of concentrated hydrochloric acid. Pour 4 g of potassium iodide(KI) into another beaker, add a little water and stir until KI is completely dissolved. Mix the two solutions, Observe the formation of a dark orange solution.

14


b. Anisaldehyde sulfuric acid reagent: Anisaldehyde (0.5 ml) + glacial acetic acid (10 ml)

+ Methanol (85 mL) + H2SO4 (5 mL) mixed in order. c. Natural products (NP-PEG) reagent: The plate is sprayed with 1% methanolic

diphenylboric acid β-ethylamino ester, followed by 5% ethanolic polyethylene glycol4000. d. FeCl3 reagent: To make neutral FeCl3 solution, first add ammonium hydroxide solution

to FeCl3.The formation of precipitate is observed. Now add FeCl3 solution again to dissolve the ppt. a clear solution is formed. This neutral FeCl3 solution is used for phenolic (-OH) test. 5.5. Extraction method 850 g dried powder of Carica papaya leaves was subjected to soxhlet extraction using 100% hexane (for defatting and to remove chlorophyll) . The weight of hexane extract was found to be 45 g. The defatted dried powder (700 g) was further extracted by soxhletion with methanol and water and 100g of defatted powder with ethanol and water in 70:30 ratio for 12 hrs. The extractive value of both the extracts was found to be 155.91 g and 21.4 g respectively. The acid base extraction method was further used for the fractionation of methanolic extract for acidification using 2N, 2% HCl used and for basification using liq. ammonia). The extractive value of crude alkaloids from methanolic extract was found to be 7 g and from the ethanolic extract was found to be 450 mg. The obtained extract was further partitioned with ethyl acetate for three time using acid base extraction technique. A schematic representation procedure of partitioning is given below.

15


Scheme 1: Schematic representation of extraction procedure.

16


Note - Same procedure used for ethanolic extract (21.4g) for acid base extraction. Finally Weight of crude alkaloids extract obtained was 500 mg. 5.6. TLC profiling of aqueous methanolic extract The TLC profiling of methanolic and ethanolic extract was done on precoated TLC plate (TLC silica gel 60 F254, 20×20) using chloroform and methanol (9 : 1) as mobile phase then TLC was also observed under UV light at short wavelength (254 nm, TLC-B) and at long wavelength (365nm, TLC-C). The TLC was derivatized with Dragendorff’s reagent and Rf values were found to be 0.48, 0.43, 0.31, 0.2, 0.1 respectively.

Figure 6: TLC profile of methanolic extract

A comparative TLC profiling of methanolic and ethanolic extract were done. Solvent system used for TLC profiling was chloroform and methanol (9:1). Reagent used to derivatize TLC was Dragendorff’s reagent. A represents derivatized TLC, B represents TLC observed in UV at 254 nm and C represents TLC observed in UV at high wavelength (365nm) of UV light.

Figure 7: Comparative TLC profiling of ethanolic and methanolic extract of Carica papaya L.

17


5.7. VLC fractionation of alkaloids rich extract Methanolic crude extract of alkaloid (7g) from Carica papaya leaves was adsorbed on 60-120 mesh size of silica gel (25g). After adsorption it was subjected to VLC fractionation where silica gel G (300g) used to pack the funnel. Fractionation/elution was done in a gradient manner using solvent combination of hexane, chloroform and methanol as 20%,40%, 60%, hexane in chloroform and chloroform in methanol as 5%, 10%, 20%, 30-80%. Each fraction of 2L volume was collected and total 14 fractions were collected and concentrated using rota vapor. The TLC profiling of all the VLC fractions was done using TLC plate (TLC Silica gel 60 F254 , 20×20) using chloroform and methanol as mobile phase(9:1). A schematic representation of VLC fractions is showing below.

Figure 8: VLC fractions of MeOH alkaloids rich extract.

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5.8. TLC profiling of VLC fractions TLC profiling of VLC fractions were done using chloroform and methanol as mobile phase (9:1) and derivatized with Dragendorff’s reagent to check the presence of alkaloids.

Figure9: TLC Profile of VLC fractionation.

In 5% methanol in chloroform VLC fraction, 1 spot showed orange color with Dragendorff’s reagent, in 10% VLC fraction, 2 spots showed orange color, in 20% VLC fraction, 2 spots showed orange color, in 30% VLC fraction, 1 spot, in 40% VLC fraction 2 spots, in 50% VLC fraction 2 spots, in 60% VLC fraction 1 spot and in 80% methanol in chloroform VLC fraction 1 spot was positive with the Dragendorff’s reagent. 5.9. Column chromatography 600 mg of the sample obtained by VLC fraction of 10 % was subjected to column chromatography where Column length × Diameter (40×3 cm) and silica gel 60-120 mesh (1.5g) for adsorption of sample (600 mg) and 230- 400 mesh for column packing (50 g)and wet method was used for the packing of column (35 g of silica gel 230-400 mesh size was taken to pack the column with continuous tapping. The adsorbed sample was loaded on the column and elution was in gradient manner where flow rate of the column was 6 ml/min and fraction volume was 30 mL. A total number of 40 fractions were collected and pooled on the basis of TLC profiling. The pooled fraction (27-34) was further subjected to column chromatography and gradient elution

19


was done using chloroform and methanol (8:2) as eluent. Various fraction was collected and fraction showing single spot on TLC were pooled and concentrated. A white amorphous powder was obtained which purity was checked using analytical HPLC and submitted for 1H NMR, 13C NMR, mass spectroscopy and melting point. A schematic representation of column fractionation shown below.

Scheme 2: A schematic representation of column chromatography

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5.9.1. TLC profile of isolated compound TLC profiling of isolated compound was done using precoated silica gel TLC plate ( TLC Silica gel 60 F254, 20×20), where solvent system used as mobile phase chloroform and methanol (7:3). The TLC was derivatized with anisaldehyde sulfuric acid reagent, and spot showed dark green color on TLC and Rf of the compound was found to be 0.42.

Figure 10: TLC profiling of isolated compound

5.9.2. Column chromatography of chloroform extract Column chromatography of VLC fraction F5 (100% CHCl3 VLC fraction; 485mg) was performed where silica gel silica gel 60-120 mesh (1.2g) was used for adsorption of sample and 230- 400 mesh size for column packing and column was packed by wet method of column packing and solvent used for packing was hexane and gradient elution was done using solvents hexane : chloroform (40% chloroform in hexane to 100% chloroform).

21


Schematic representation of chromatography process shown below.

Scheme 3: Schematic representation of column chromatography.

A total of 26 fraction were collected. Fraction 3-8 was pooled on the basis of TLC profiling and

again subjected to column chromatography and gradient elution was done using hexane and chloroform as mobile phase (40% chloroform to 60% chloroform in hexane). A total of 22 fractions were collected and fraction showing single spot (F17-18) on TLC were pooled and concentrated. TLC was done using hexane and chloroform (1 : 1) as mobile phase.

22


6. RESULTS AND DISCUSSION 6.1. Compound -1 A comparative TLC profiling of isolated compound and β-sitosterol as standard showed both spots have same Rf value (0.31).

Figure 11: Comparative TLC profiling of β-sitosterol as standard and isolated compound

TLC profile revealed that isolated compound is β-sitosterol (17-(5-Ethyl-6-methylheptan-2-yl)10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol, C29H50O). It is a white waxy powder with melting point 138ºC and reported mp is 136ºC, total amount of isolated β-sitosterol isolated is 11 mg. 6.2. Compound 2: IPGJ-12 It is a white amorphous powder, soluble in methanol, Rf value was found to be 0.42 using solvent chloroform and methanol (7:3) as mobile phase and melting point was found to be 118 ºC. 1H NMR of compound,

13

C NMR with DEPT-135, mass spectrum and HPLC profile of the

compound shown below.

23


Figure 12: 1H NMR spectrum of compound (IPGJ-12)

24


Figure 13:

13

C NMR spectrum of compound (IPGJ-12)

Figure 14: DEPT-135 carbon NMR spectrum of compound (IPGJ-12)

25


svir

ipgj-12-a 2 (0.505) Cm (1:4) 100

Scan ES8.14e5

134

%

541

406

271

0 100

150

200

250

300

350

400

450

500

550

600

650

700

Figure 15: Direct mass spectrum of isolated compound (IPGJ-12)

26

750

m/z 800


6.2.1. HPLC Chromatography HPLC Chromatography of Sample (IPGJ-12) was done and for Sample preparation, 1 mg of the

sample was dissolved properly in MeOH and sample was passed through .45µm filter, the injection volume was 5µl where Chromatographic system was shimadzu LC-10ATVP system (shimadzu corp, Kyoto, Japan) consisting of model LC-10Atvp fitted with SIL- 20AC auto sampler and SPD-M10A VP photodiode array detector (PDA). Column Sunfire C-18, 4.6 x 150 mm used in HPLC and MeOH : Water (8:2) used as mobile phase in isocratic manner. Detection wavelength for the compound was 259 nm and retention time was found to be 2.605. The Purity index was found to be 99.84%

Figure 16: HPLC chromatogram of isolated compound (IPGJ- 12)

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7. SUMMARY Carica papaya L. plant was selected for isolation and characterization of secondary metabolites from the methanolic extract of leaves. Methanolic extract was prepared by using soxhlet apparatus and partitioned with ethyl acetate and acid base extraction for isolation of alkaloids, and other secondary metabolites. Two compounds were isolated, compound one found to be βsitosterol and characterization of second compound is under process.

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―New

Macrocyclic

Piperideine

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papaya

leaves:

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