International Journal of PHARMACEUTICAL AND BIOMEDICAL

RESEARCH

Research article

Phytochemical investigation of the insulin plant “Costus pictus” D. Don C.T.Shiny*, Anuj Saxena, Sharad Prakash Gupta

Department of Botany, Sacred Heart Degree College, Sitapur-261001, Uttar Pradesh, India

Received: 10 Apr 2013 / Revised: 19 Apr 2013 / Accepted: 20 Apr 2013 / Online publication: 26 Apr 2013

ABSTRACT

Costus pictus D. Don (Spiral ginger) commonly known as ‘Insulin plant’ was introduced from Mexico to India (Kerala) very recently. The people in Kerala used to consume the fresh raw leaves for its anti-diabetic activity. The hypoglycaemic activity of Costus pictus is mainly because of secondary metabolites. Despite the preliminary studies the detailed phytochemical investigation has not been reported so far. Therefore, the leaves stem and rhizome extracts of Costus pictus in different solvents (hexane, ethyl acetate, methanol and water) were subjected to phytochemical studies. Even though there was negligible difference in the presence of chemical constituents in all 24 extracts of three samples; the methanol extract of leaves has shown maximum number and concentration of secondary metabolites based on phytochemical and TLC tests. Further analysis such as Column chromatography, HPLC and GC-MS of leaf methanol extract has revealed the presence of a glycoside compound similar to a reference compound, β-L-Arabinopyranose methyl glycoside and that might be the inducer molecule of its antidiabetic property.

Key words: Costaceae, Secondary metabolites, Thin layer chromatography, High performance liquid chromatography, GC-MS, Column chromatography, Glycoside, β-L-Arabinopyranose methyl glycoside.

1. INTRODUCTION

The phytochemical analysis of Costus pictus has been conducted by George et al., [1] and the saponified extracts (ether and acid fractions) were analyzed using GC-MS for the identification of its chemical constituents, and a total of 18 chemical compounds were identified from the plant leaves. C. pictus is also rich in antioxidants [2]. Jayasri et al. [3] estimated the presence of trace elements in C. pictus and showed that the leaves and rhizomes contain appreciable amounts of various elements, such as K, Ca, Cr, Mn, Cu, and Zn, which may be responsible for potentiating insulin action. C. pictus leaf contains various phytochemicals like alkaloids, glycosides, carbohydrates, saponins, proteins and phenols [4,5]. Jothivel et al. [6] carried out TLC separation to identify the active constituents responsible for the antidiabetic activity by using specific solvent system and they presumed that pentacyclic triterpene compounds such as β-amyrin might be

the active principle contributing to the antidiabetic activity. Shilpa et al. [7] isolated methyl tetracosanate from bioactivity-guided purification of methanolic extracts of C. pictus leaves which showed an optimum glucose uptake at 1ng/mL in 3T3-L1 adipocytes. Beena and Reddy (2010) [8] analysed the volatile constituents of C. pictus stems, leaves and rhizomes and palmitic acid was found to be the major component in the stem, leaf and rhizome.

In the context of the above, an intense research work is essential in the perspective of findings the responsible phytochemical(s) for the antidiabetic activity. Therefore, in the present research work, the plant material was extracted in different organic solvents; consecutively chromatographic analysis of the extracts was done, in order to identify the active principle(s) for proper exploitation of the plant in antidiabetic therapeutics.

2. MATERIALS AND METHODS

The plants were cultivated and identified by Dr. Santhosh Nampy, Department of Botany, St. Joseph’s College, Devagiri, University of Calicut and herbarium specimen has

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Int J Pharm Biomed Res 2013, 4(2), 97-104

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C.T.Shiny et al, Int J Pharm Biomed Res 2013, 4(2), 97-104

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been submitted to Botany Department (Ref. No. SJC/BOT/RES-EXT/1/2012). Leaf, stem and rhizome portions of one year old C. pictus plants were separately harvested, washed, chopped and dried at room temperature. The well dried materials were ground into fine powder and kept in air tighter zip bags separately (Fig.1 &Table 1).

Fig.1.Dried powder of Costus pictus Table 1 Effect of leaf, stem and rhizome of Costus pictus on drying Materials Fresh weight Dry weight Weight loss after drying Leaves 10kg 1.50 kg 85.0% Stem 10kg 1.25 kg 87.5% Rhizome 10kg 1.10kg 89.0%

2.1. Phytochemical screening

Phytochemical screening was performed using appropriate procedures [9- 11] involving extraction, isolation and identification of secondary metabolites.

2.1.1. Extraction of the plant material

There are various methods of extraction [12]; two types of extraction methods have been tried in the present study (cold maceration and hot continuous extraction or soxhlet extraction) to check the presence of thermo labile components in C. pictus as they may be lost in elevated temperature.

(A) Cold maceration

500g of air dried, powdered material (rhizome, stem and leaves) was separately macerated with hexane, ethyl acetate,

methanol and water (Aqueous) successively for 48 hours with occasional stirring [9]. The mixture was then filtered after 48 hours. The filtrates were evaporated to dryness using a rotary evaporator at 40°C under reduced pressure.

(B) Hot continuous extraction

500g of air dried and powdered material (leaves, stem and rhizome) was being extracted separately with hexane, ethyl acetate, methanol and water (Aqueous) successively in a soxhlet apparatus for 6 hours. The extract was concentrated using rotary evaporator at 40°C under reduced pressure.

2.1.2. Separation of the major phytochemicals

(A) Phytochemical tests

1g of the plant extract was dissolved in 100mL of the respective mother solvents to obtain a stock of 1% concentration (w/v). The extracts thus obtained were subjected to preliminary phytochemical tests. The qualitative phytochemical tests for steroids, triterpenoids, glycosides, phenols, alkaloids, quinones, coumarins, furanoids, flavonoids and tannins were performed on different extracts (Fig.2).

Fig.2. Graphical presentation of the phytochemical analysis of Costus pictus

(B) TLC (Thin Layer Chromatography) of extracts

All the 24 extracts prepared through two different extraction methods were selected for TLC analysis. Pre-coated TLC plates (20 x 20cm) were used as the stationary phase. Different mobile phases were tried and finally Chloroform: Ethyl Acetate: Methanol: Benzene in the ratio of

C.T.Shiny et al, Int J Pharm Biomed Res 2013, 4(2), 97-104

99

70: 4: 8: 24 was standardized as the best mobile phase to obtain clear spots. Freshly prepared p-anisaldehyde reagent was used as a spraying reagent. The samples (10 µL, 20µL) were loaded on TLC plates at equal distance.

Rf values were calculated [13] after the scanning of the chromatograph. The result of phytochemical tests and TLC analyses indicated the presence of similar secondary metabolites in various extracts from three types of samples. Keeping in view of these preliminary results, methanol extract of leaves was taken for further analyses as this gave the maximum yield in the extraction.

(C) HPLC (High Performance Liquid Chromatography) of the methanol extract

HPLC was performed on a Shimadzu model LC 10AD equipped with a detector SPD10A. The methanolic extract (20μL) was subjected to HPLC analysis under the following operating conditions. Column – C18 LiChroCART-merck (4.6 x 30cm), Stationary phase - Octa decyl silane, Mobile phase - Acetonitrile: water (60:40, v/v), Flow rate - 1mL/min, Detector – UV, Wave length - 210nm and Flow rate - 1mL/min, Detector – UV.

Table 2 The colour and percentage yield of 24 extracts of Costus pictus Extraction solvents Plant material Cold extraction method Hot extraction method

Colour Yield (g) % yield Colour Yield (g) %yieldHexane Leaves dark green 24.89 4.978 dark green 4.61 0.93

Stem dark green 1.73 0.346 dark green 2.23 0.45 Rhizome pale yellow 2.83 0.566 pale yellow 2.05 0.41

Ethyl Acetate Leaves dark green 25.81 5.16 dark green 5.76 1.15 Stem bright yellow 21.67 4.34 yellow 2.54 0.51 Rhizome pale yellow 5.73 1.15 pale yellow 2.31 0.47

Methanol Leaves dark green 84.5 16.9 dark green 11.59 2.32 Stem bright yellow 40.34 6.07 yellow 4.69 0.94 Rhizome pale yellow 7.91 1.60 pale yellow 3.47 0.70

Aqueous Leaves bright yellow 29.14 5.83 yellow 6.46 1.30 Stem yellow 17.35 3.47 yellow 4.38 0.87 Rhizome pale yellow 6.30 1.26 pale yellow 4.03 0.81

Table 3 Preliminary phytochemical tests of different extracts of leaves, stem and rhizomes of Costus pictus obtained by maceration Extraction solvents Plant material Phytochemicals/ Secondary metabolites

Steroid Triterpenoid Alkaloid Phenol Glycoside Quinones Coumarins Flavanoid Furanoid TanninHexane Leaves + + + + + + + + - -

Stem + + + + + - + + - - Rhizome + + + - + - - + - -

Ethyl Acetate Leaves + ++ + + ++ + + + - - Stem + ++ + + ++ - + + - - Rhizome + ++ + - ++ - - + - -

Methanol Leaves + ++ ++ ++ ++ + + ++ - - Stem + ++ ++ ++ ++ - + ++ - - Rhizome + ++ ++ - ++ - - ++ - -

Aqueous Leaves + + + + + + + + - - Stem + + + + + - + + - - Rhizome + + + - + - - + - -

(+) Presence, (++) high concentration, (-) Absence Table 4 Preliminary phytochemical tests of different extracts of leaves, stem and rhizomes of Costus pictus obtained by hot continuous extraction Extraction solvents Plant material Phytochemicals/ Secondary metabolites

Steroid Triterpenoid Alkaloid Phenol Glycoside Quinones Coumarins Flavanoid Furanoid TanninHexane Leaves + + + + + + + + - -

Stem + + + + + - + + - - Rhizome + + + - + - - + - -

Ethyl Acetate Leaves + ++ + + ++ + + + - - Stem + ++ + + ++ - + + - - Rhizome + ++ + - ++ - - + - -

Methanol Leaves + ++ ++ ++ ++ + + ++ - - Stem + ++ ++ ++ ++ - + ++ - - Rhizome + ++ ++ - ++ - - ++ - -

Aqueous Leaves + + + + + + + + - - Stem + + + + + - + + - - Rhizome + + + - + - - + - -

(+) Presence, (++) high concentration, (-) Absence

C.T.Shiny et al, Int J Pharm Biomed Res 2013, 4(2), 97-104

100

Fig.3. TLC of various extracts of different concentrations (A)-10µL, (B)-20µL Table 5 Rf values of 9 spots of TLC of 12 crude extracts (Maceration) of Costus pictus Spots Type of extracts

HL H S HR EaL EaS EaR ML MS MR AqL AqS AqR 1 0.0882 0.0873 0.0883 0.0885 0.0875 0.0878 0.0879 0.0884 0.0881 0.0882 0.0873 0.08682 0.1588 0.1467 0.1571 0.1566 0.1455 0.1464 0.1512 0.1563 0.1567 0.1581 0.1536 0.15863 0.3235 0.3342 0.3353 0.3242 0.3521 0.3532 0.3351 0.3461 0.3205 0.3156 0.3222 0.32404 0.4706 0.4657 0.4745 0.4651 0.4712 0.4723 0.4733 0.4710 0.4722 0.4731 0.4724 0.47125 0.6765 0.6544 0.6621 0.6546 0.6645 0.6761 0.6753 0.6771 0.6683 0.6739 0.6698 0.67276 0.7059 0.7132 0. 7121 0.7061 0.7044 0.7071 0.7017 0.7063 0.7055 0.7063 0.7057 0.70487 0.7648 0.7712 0.7646 0.7655 0.7743 0.7723 0.7664 0.7711 0.7710 0.7646 0.7657 0.76588 0.8235 0.8331 0.8324 0.8238 0.8321 0.8322 0.8256 0.8251 0.8254 0.8326 0.8250 0.82419 0.8529 0.8621 0.8630 0.8541 0.8516 0.8611 0.8567 0.8548 0.8577 0.8542 0.8613 0.8545H-Hexane, Ea-Ethyl acetate, M-Methanol, Aq-Aqueous, L-Leaf, S-Stem, R-Rhizome

Table 6 Rf values of 9 spots of TLC of 12 crude extracts (Hot continuous extraction) of Costus pictus Spots Type of extracts

HL H S HR EaL EaS EaR ML MS MR AqL AqS AqR 1 0.0890 0.0885 0.0879 0.0883 0.0875 0.0873 0.0882 0.0881 0.0879 0.0884 0.0879 0.08862 0.1584 0.1567 0.1581 0.1561 0.1495 0.1564 0.1502 0.1560 0.1566 0.1573 0.1546 0.15833 0.3243 0.3342 0.3353 0.3242 0.3521 0.3532 0.3351 0.3461 0.3205 0.3156 0.3222 0.32404 0.4705 0.4677 0.4715 0.4631 0.4722 0.4703 0.4740 0.4720 0.4725 0.4732 0.4723 0.47325 0.6754 0.6564 0.6634 0.6543 0.6625 0.6763 0.6773 0.6731 0.6724 0.6729 0.6699 0.67576 0.7122 0.7021 0. 7111 0.7082 0.7044 0.7068 0.7097 0.7089 0.7132 0.7088 0.7090 0.71187 0.7651 0.7710 0.7648 0.7681 0.7714 0.7731 0.7697 0.7701 0.7730 0.7647 0.7687 0.76948 0.8230 0.8321 0.8324 0.8231 0.8221 0.8312 0.8216 0.8230 0.8214 0.8320 0.8240 0.82369 0.8531 0.8634 0.8631 0.8532 0.8516 0.8510 0.8537 0.8546 0.8570 0.8582 0.8527 0.8571H-Hexane, Ea-Ethyl acetate, M-Methanol, Aq-Aqueous, L-Leaf, S-Stem, R-Rhizome

(D) Column chromatography

500mg of methanolic extract was separated into various fractions using column chromatography. The column (a vertical glass tube of 50 x 2cm) with a stopper was packed by wet method using 75g of silica gel (stationary phase) and hexane, 500mg of the extract was loaded by using pure hexane (mobile phase, non-polar) as the solvent. For the elucidation of components, the polarity of the solvent (mobile

phase) was increased using a combinations of hexane : ethyl acetate in the ratio of 9:1, 7:3, 5:5, 3:7, 1:9 successively. Similarly the column was run over ethyl acetate: ethanol and ethanol: methanol combinations in order to increase polarity.

A total of 5 fractions (1, 2, 3, 4, 5) were obtained through column chromatography. Each fraction was collected separately, concentrated under reduced pressure in a rotary evaporator and analyzed by TLC using chloroform: ethyl acetate: methanol: benzene (70: 4: 8: 24) as the mobile phase.

(A) (B)

anfrfrfr

2.

MSQ.5stphte30th

3.

3.

hamsolev29diex217sosiwTboan

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prglexexphreTPexqutr(T

The plates nd then sprayractions collecraction was apraction was an

.1.3. Identifica

Gas liquid MS) analysis

himadzu ModQP 5000A mas5mm) packedtationary phahase (carrier emperature a) 0°C – 250°Chrough the atta

. RESULTS

.1. Extraction

The 24 exave shown c

maceration, tholvents was foeaf extracts ths 4.61g; EA:9.14g vs 6.46isparity in thexcept in the e.23g; EA: 217.35g vs 4.38olvents and timilar. In abo

was obtained fThe maximum

oth extractionnd hexane (lea

.2. Phytochem

The prelimresence of lycosides, quxtracts of leafxtracts furnishytochemical esults in respe

The furanoids henols, quinoxtracts. All suinones. Theriterpenoids aTable 3 & Tab

were observeyed with aniscted through ppeared as thenalyzed using

ation of the m

chromatograwas perform

del GC-17A ss spectrometd with 100%se and Heliu

gas). 1μL Initial – 30°C

C for 10 minuached Mass sp

n of plant mate

xtracts yieldedconsiderable e yield of exound to be 4 through hot co 25.81g vs 56g). Both exte yield of stemextracts prepa1.67g vs 2.54g). The yield two extractioove two extracfrom the leave

m extract yieldn procedures foast polar solve

mical tests

minary phytosteroids, tri

uinones, coumf, stem and rhshed essentia

tests conduect of all 24 ex

and tannins ones and coustem extractse presence oand glycosideble 4).

C.

ed under UV saldehyde reacolumn chrom

e major compoHPLC and GC

major compoun

aphy – Mass med on the equipped wit

ter. The Capil% dimethyl pum (0.6mL/m

of sample iC for 1 minuteutes. The specpectrometer a

erial

d by two exdifferences (

xtracts from leto 7 fold grea

ontinuous extr5.76g; Met: 8traction procem extracts usiared with hex4g; Met: 40.of rhizome exn proceduresction procedues followed byd was obtainefollowed by aqent).

ochemical teiterpenoids,

marins and flahizome portioally same reucted, eight txtracts of leafwere lacking

umarins were also gave nof alkaloids, es was obser

.T.Shiny et al, Int

light at 302nmagent. Amongmatography, tonent. TherefoCMS.

nd

spectrometry2nd fraction

th Shimadzu lary column (

polysiloxang min) used as m

injected at ce (b) Programctrum was obs the detector.

xtraction proc(Table 2). Ineaves using v

ater than the yraction (Hex 84.5g vs 11.5edures also sing various so

xane (Hex: 1.734g vs 4.69g

xtracts using vs was more oures maximumy stem and rhed with methaqueous, ethyl a

ests indicatealkaloids, phavanoids in vons of C. pictuesults. Amontests gave pf, stem and rh

g in all the exabsent in rh

negative resuphenols, st

rved in all e

t J Pharm Biomed

m [13] g the 5 the 2nd ore, 2nd

y (GC-using

model (30m x as the mobile column

mming - btained .

cedures n cold various yield of 24.89g g; Aq:

showed olvents 73g vs g; Aq: various or less

m yield hizome. anol in acetate

ed the henols, various us. All ng ten positive hizome. xtracts. hizome ults for teroids, extracts

3.3

proTLspexexprofirab

anwatyponexmaphfur

3.4

exshpeshof Thwa

d Res 2013, 4(2),

3. TLC of extr

TLC of vocedures gaveLC showed thots were not

xtracts gave simxtracts of leafofiles of separst two spots sent in extractThe results

nd TLC (Tableay of major chpes of sample

ne type of extrxtract of leafaximum yiel

hytochemicals rther detailed

4. HPLC of th

The result oxtract has beeows the prese

eaks were majoown similar p

f the two peakhe percent conas 39.93 and 3

Fig.4. HP

97-104

racts

various extrae similar patthat 9 spots w

so apparent.milar Rf valuf, stem and aration by wapresent in le

ts of rhizome.of phytochem

e 5 & Table 6hemical consties. Hence forract and one saf (macerationld (16.9%),

(Table 3 & Tinvestigations

he crude metha

of HPLC anaen depicted ence of 15 peaor. The TLC rpattern of banks contributed ntribution on 30.70%, respe

PLC of methanoli

acts of usintern of spots

were clearly de. The major

ues (Table 5 &rhizome por

ay of differeneaf and stem . mical tests (T6) have also situents in all tr further chemample was us

n) as shown and higher

Table 4), and s.

anolic leaf ext

alysis of crudin the chrom

aks. Among thresult of crudends (Fig.5). T 42.27 and 39height basis

ectively.

ic leaf extract of

ng two extra(Fig.3). Resuetectable and spots found

& Table 6). Vartions gave snt spots, exce

extracts that

Table 3 & Tabshown similarthe extracts ofmical analysised. The methain Table 2 concentratio

it was selecte

tract

de methanolicmatograph (Fhese, the 4th ae methanol ha

The percent on9.80% respectof these two

Costus pictus

101

action ults of

other in all arious imilar pt for were

ble 4) ity by f three s only anolic

gave on of ed for

c leaf Fig.4); and 5th as also n area tively. peaks

Fig.5.TLC of m

Fig.6.Fract

Fig.7. HPL

(A)

methanol crude ext(A)-10

tions (5) of colum

C of 2nd fraction

C.

tracts of leaf in dµL, (B)-20µL

mn chromatograph

following column

(B)

.T.Shiny et al, Int

ifferent concentra

hy and their TLC

n chromatograph

t J Pharm Biomed

ations

C

y

3.5

cosoeluHeEthEthEthEth

acthe

3.6

antimtintheres

3.7

anspin loodato Th

3.8sp

Mthe+ =givfrawahapacorefpeThRe16Iso17

d Res 2013, 4(2),

5. Column chr

Five fractionlumn chromalvents used foution was carrexane: Ethyl Ahyl Acetate: Mhyl Acetate: Mhyl Acetate: Mhyl Acetate: M

The major etate: methane other fractio

6. HPLC of th

High perfonalysis of 2nd fme of the majny and appeare area and heispectively.

7. TLC of the2

The thin laynd the same wots are visiblecolour when

oked brown uark after spray

confirm the his was also de

8. GC-MS (Gaectrometry)

Gas liquid S) spectrum (e reference co= 165 (Fig.9Aven in Fig.9b,agmentation pas found that ad somewhat articularly up nclusively thference stand

eaks are differehe major fragmeference comp65 (MH+) olated compou71

97-104

romatography

ns (1, 2, 3, 4, atography (Figor the isolationried out as follAcetate (1: 9) Methanol (7: 3Methanol (5: 5Methanol (3: 7Methanol (1: 9

band was enol 7:3 v/v. Tons were green

he 2nd fraction

ormance liqufraction (Fig.7jor peak was red insignificaight of the ma

2nd fraction

yer chromatowere visible ue. The 4th spotn compared t

under UV lightying with amm

presence of etected with a

as liquid chro

chromatograp(Fig.8, Fig.9Aompound had A). The structu, and the samppattern as thatthe compoundmatching witto m/z 116;

hat both theard are the sent. mentation ionspound: m/z at

und: m/z at 4

y

and 5) were g.6). Based onn of various flows: - first fraction

3) - second fra5) - third fract7) - fourth frac9) - fifth fractiluted with a

The first fractnish yellow or

uid chromat7) showed 7 p

6.89 min. Alant. The perceajor peak wer

graphy (TLCunder UV light in the TLC wto other spott at 302nm wamonia. These glycoside in

anthrone reage

matography –

phy – Mass A & Fig.9B) cl

a molecular wure of the refeple gave somet of the referd isolated froth that of refstill it is ve

e isolated cosame as the p

s seen are as f43, 55, 69, 81

43, 57, 73, 83

obtained follon the polarity fractions, sequ

n (1) action (2a, 2b)tion (3) ction (4)ion (5)

a mixture of tion was greer yellow in col

tographic (Heaks. The retell other peaksent contributiore 99.75 and 9

C) revealed 6 ht. In Fig.6, owas more yellts. The sameavelength. It ttwo results hthe crude sa

ent.

– Mass

spectrometry learly indicateweight of 164 erence compouewhat similarrence compoum C. pictus eference compery difficult tompound andparent (MH)

follows: 1, 96, 116, 133

, 97, 116, 133

102

owing of the

uential

)

ethyl n and lour.

HPLC) ention s were ons of 98.84,

spots only 5 owish

e spot turned helped ample.

(GC-es that (MH) und is r same und. It extract pound, to say d the + ion

3, and

3, and

Fi

ig.9A.Fragmentat

Fig.9B.Fragmen

Fig.10.Structur

Fig.8. Spectr

tion pattern of sam

ntation pattern of (reference com

HO

OH3C

re of β-L-arabinoco

C.

ral graph of GC-M

mple obtained du

f β-L-arabinopyrampound) by GC-M

O

OH

OH

opyranose methylmpound)

.T.Shiny et al, Int

MS

uring GC-MS ana

anose methyl glycMS

H

l glycoside (refere

t J Pharm Biomed

alysis

coside

ence

ideHoide

4.

exusstuof difrepinvinf

mesoxonfiltfoumedopiccocarprephphto in

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d Res 2013, 4(2),

It was concentified as β-Lowever, furtheentity.

DISCUSSIO

The findingsxplore the antie, and especiudies have beef C. pictus afferent laboratport may be vestigation oformation ava

Comparativeethod was higxhlet method.

ne likely reasotering in theund in the lethanol. The t

one to check thctus. The therntinuous extrried out at eliminary ph

henols, etc. revhytochemicals

indicate the gC. pictus. The presen

ncentration ind not show an

hytochemical nd TLC (Tablowed similari

Methanolic umber as wellhis result is m,6,14]. The m

xhibit maximue same is repoported similaostus have alsethanolic extr

ecided to procf leaves (murification anmpounds. The crude m

he HPLC chroeaks (4thand 5vestigated. Tmpounds in t

97-104

cluded to beL- arabinopyraer work is re

ON

s of this studyidiabetic potenially in India.en reported onand investigatories none ofthe first info

f C. pictus ailable on its pe yield of extrgher than that. The reason oon may be rele maceration leaf extract two kinds of he presence ofrmo labile comtraction meth

an elevatedhytochemical vealed similarin the two typ

general absen

nce of secon various extrny major diffeanalyses. Thele 3 & Tableity in the chemleaf extract ol as concentr

matching with methanolic leum and pronoorted by the auar finding [6,so been reporract [17]. Baseed for furthe

maceration) wnd hopefully

methanolic leafomatogram sh5th) obtained bThis indicatedthe methanoli

e a glycosidanose methyl quired to con

y should pavential of C. pic. Even thoughn phytochemiations are alf them is muchormation on even though

phytochemistryracts obtainedt of the extracof this differenlated to less c

technique. Hprepared by extraction prof thermo labilmponents mayhod because d temperatur

qualitative trity by way opes of extract

nce of thermo

ondary metabracts of the s

erence (Table e results of pe 4, Fig.3) o

mical constitueof C. pictus

ration of secothe findings

eaf extract wounced antidiauthor. Previou7,14-16]. Therted to have ssed on these oer analysis witwith the aimy with the

f extract was showed 15 peaby HPLC anad the presenic leaf extract

de and tentaglycoside (Fi

nclusively pro

e the way to fuctus for worldh a few peripical constituenso in progreh informativethe phytochea few perip

y. d by the macects prepared bnce could be mclarification thHighest yield

maceration ocedures were componentsy be lost in th

the extractire [12]. Howtests for ste

of presence of s. This would labile constit

bolites and same plant sam3 & Table 4)

phytochemicalof 24 extractsents. showed max

ondary metaboof previous st

was also showabetic activityus studies have other specisimilar effectsobservations ith methanol em of separ

identificatio

subjected to Haks. The two alysis were fu

nce of two t. The contrib

103

atively ig.9b). ove its

further d-wide pheral nts [6] ess in e. This emical pheral

eration by the many; hough d was

using e also s in C. he hot on is

wever, eroids, f these d seem tuents

their mples in the l tests s also

ximum olites. tudies wn to y, and ve also ies of s with it was extract ration, on of

HPLC. major

further major bution

C.T.Shiny et al, Int J Pharm Biomed Res 2013, 4(2), 97-104

104

of these two peaks was 42.27% and 39.80%, respectively and these values indicate that 82.07 % of the extract was due to these two compounds. The remaining 17.93% of the extract was due to 13 other minor compounds. Further work was not done on these minor constituents, and studies were concentrated on the two major constituents.

Column chromatography [9] separated a total of 5 fractions (bands) and the second band was the major fraction (Fig.6); this result matches with the results of HPLC analysis of the crude methanolic leaf extract. The 4th and 5th peaks in the HPLC, 4thand 5th bands in the TLC analysis (Fig.4 & Fig.5), and 2a and 2b fractions following column chromatography (Fig.6) would appear to be the same compounds. The HPLC chromatogram of 2nd fraction showed one major peak (99.75% peak area) with 6 other peaks of very minor nature ion; among the 6 spots on TLC (Fig.6), visualized under UV light, the 4th one was intense, concentrated and clear compared to the other 5 spots. This spot looked brown under UV light at 302nm wavelength and it turned dark when it was sprayed with ammonia. These two tests done on TLC are suggestive to the presence of a glycoside [9]. Therefore, it was concluded that the major phytochemical found in the crude methanolic leaf extract is one of several glycosides. The presence of glycoside was also detected in the preliminary phytochemical qualitative tests with anthrone reagent. The presence of spirostanol glycoside and furostanol glycoside has been reported in C. speciosus [18,19]. β-Sitosteryl-d-glucoside isolated from the bark of Ficus religiosa has shown hypoglycemic activity in normal rabbits [20]. Nath (1943) [21] reported the antidiabetic effect of Scoparia dulcis, obtained as glycoside named ammelin from fresh plants which relieved other ailments accompanying diabetes, such as pyorrhea, eye troubles, joint pain, susceptibility to cold, etc., within a short period.

The same second fraction of column chromatography following HPLC purification was subjected to GC-MS [22] and the spectral profile of the sample and the reference (marker) compound (Fig.9A & Fig.9B) showed some similarity in their molecular fragmentation at least up to M+ 116. Β-L-arabinopyranose methyl glycoside was used as reference compound (Fig.10). Based on some similarity in the fragmentation pattern of the sample with the reference compound, it would seem reasonable to say that the glycoside in the sample could be β-L-arabinopyranose methyl glycoside.

However, the identification at the moment can best be said as highly tentative and much more work is required to prove its identity conclusively. This GC-MS data may helpful for further analyses by FTIR & NMR that are required for detecting functional groups present in the compound that will help in identifying the compound. This compound would also seem to have antidiabetic activity, and it is extracted in the

methanol fraction of the leaf extract. Previous studies have also reported maximum and pronounced antidiabetic activity of methanolic leaf extract. This report would be helpful for the future investigations on various potentialities of C. pictus.

5. CONCLUSIONS

Based on the above findings, it can be concluded that the antidiabetic activity of C. pictus could be due to the presence of a phytochemical, β- L- Arabinopyranose methyl glycoside in the plant. However, the above mentioned active constituent has to be isolated, characterized and evaluated for antidiabetic activity in comparison with reference compound. To the best of our knowledge, this is the first report on phytochemical investigation of C. pictus even though a few peripheral information available on its phytochemistry.

ACKNOWLEDGEMENTS

This work was supported by INSA (Indian National Science Academy). The great contribution of Dr. LMS Palni (GBPIHED) and Dr. Neelam Sangwan (CIMAP) in the experimental designing interpretation of data is gratefully acknowledged.

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