MM0900108 Jour. Myan. Acad. Arts & Sc 2005 Vol. HI. No. 4(ii) Botany Phytoehemical Studies OH Momordica spp. Linn, and Extraction and Isolation of Charantin from the fruit of M.charantia L. Sanda Hlaing*, Htin Aun fc Kyaw" Abstract The genus Momordica belong to the family Cucurbitaceae. In Union of Myanmar 5-species of the genus Momordica was found Preliminary phytochemical test was carried out on the fruits of 5-species. Extraction and isolation of the active steriodal glucoside charantin compound was done on the fruit of M.charantia L. The compound charantin was confirmed by thin layer chromatography, melting point, and U.V, IR spectroscopic methods. Introduction The present era. can not ignore the important role of plant kingdom in solving the human health problems. Most of the countries have been explored and disseminated the traditional medicine knowledge of the plants so that directly or basically useful information could be postulated for the effective drug development. Myanmar has a long history of health care system by medicinal plants as a national heritage. For promotion and development of traditional medicine various categories of research work such as (a) identification of plants (b) chemical analysis (c) bio-activity tests are needed. In the present research work the genus Momordica L. is selected because it is famous in Myanmar as foods and also as medicine. Now a day, chemical compound charantin which is isolated from the fruit of M.charantia L. showed the activity on diabetes, and other compounds present are also active on tumor and HIV infections. * Lecturer, Department of Botany, University of Dawei, Dawei ** Deputy Director, Pharmaceutical Research Dept., Myanma Scientific & Technological Research Department
Phytoehemical Studies OH Momordica spp. Linn, andExtraction and Isolation of Charantin from the fruit of
M.charantia L.Sanda Hlaing*, Htin Aunfc Kyaw"
Abstract
The genus Momordica belong to the family Cucurbitaceae. In Union ofMyanmar 5-species of the genus Momordica was found Preliminaryphytochemical test was carried out on the fruits of 5-species. Extraction andisolation of the active steriodal glucoside charantin compound was done onthe fruit of M.charantia L. The compound charantin was confirmed by thinlayer chromatography, melting point, and U.V, IR spectroscopic methods.
Introduction
The present era. can not ignore the important role of plant kingdom insolving the human health problems. Most of the countries have been exploredand disseminated the traditional medicine knowledge of the plants so thatdirectly or basically useful information could be postulated for the effectivedrug development.
Myanmar has a long history of health care system by medicinal plantsas a national heritage.
For promotion and development of traditional medicine variouscategories of research work such as (a) identification of plants (b) chemicalanalysis (c) bio-activity tests are needed.
In the present research work the genus Momordica L. is selectedbecause it is famous in Myanmar as foods and also as medicine.
Now a day, chemical compound charantin which is isolated from thefruit of M.charantia L. showed the activity on diabetes, and other compoundspresent are also active on tumor and HIV infections.
* Lecturer, Department of Botany, University of Dawei, Dawei** Deputy Director, Pharmaceutical Research Dept., Myanma Scientific &
Technological Research Department
226 Jour. My an. Acad. Am & Sc. 2005 Vol. in. No. 4(ii) Botany
Materials and Methods
In this research work, plants material were collected from Yangon andBago division. The collected specimens were identified at BotanyDepartment, Yangon University and chemical studies were carried out at theLaboratory of Pharmaceutical Research Department, Myanmar Scientific &Technological Research Department. The commercial grade reagents fromBDH, MERCK, etc. were used except ethanol and petroleum ether (b.p. 60-80 C) which are local products. These local reagents were distilled for twotimes before use.
Chemical Studies
Preliminary Fhytochemical examination of the fruits of genus MomordicaL. species
Fresh unripe fruits of M.chararitia L, M.dioica Roxb., M.subangulataBlume., M. cochinchinensis Spreng. and M.muricata L were collected fromYangon & Bago division and dried in the hot air oven at the temperature of50 C. Dried material were grinded to get powder and stored in an air tightcontainers.
Preliminary phytochemical examination for the fruits was carried out(Central Council of Research in Unani Medicine 1987, Trease and Evens1980). •
Test for Alkaloids
1 g of powdered plant material was boiled with dilute hydrochloricacid and filtered. The filtrate was subsequently divided into three portions andtested with alkaloid reagents, viz Mayer's reagent, modified DragendorfPsreagent and Sodium picrate solution. Treatment with the above-mentionedalkaloid reagents furnished alkaloid precipitate, indicating the presence ofalkaloids in the plant material'
Test for Glycosides
1 g of powdered plant material were boiled with distilled water forabout 10 minutes, allowed to cool and filtered. The filtrate was treated with 10
JO ;SLJ.
Jour. Myan. Acad. Arts & Sc 2005 Vol. III. No. 4(ii) Botany 227
% lead acetate solution. Precipitation took place on the addition of the reagentindicating the presence of glycosides in the plant material.
Test for Saponin Glycosides
0.5 g powdered plant material was introduced into a test tube andshaken vigorously with distilled water for a few minutes. Marked frothingwhich lasted for about half an hour took place, indicating the presence ofsaponin glycosides.
Test for Cyanogenic Glyccsides
1 g of powdered plant material was mixed with distilled water andintroduced into a test tube. A few^drops of concentrated sulphuric acid wasadded and sodium picrate paper trapped in a neck with a cork. Sodium picratepaper did not change in colour brick-red, indicating the absence of cyanogenicglycosides.
Test for Reducing Sugar
1 g of powdered plant material was boiled with dilute sulphuric andfiltered. The filtrate was then neutralized with dilute sodium hydroxidesolution. When the resulting solution was treated with Benedict's solution, itfurnished brick-red precipitates, indicating the presence of a reducing sugar.
Test for Steroids
Tne powdered plant material 1 g was extracted with benzene and thesolvent was removed by distillation under reduced pressure. When thebenzene extract was treated with acetic anhydride and concentrated sulphuricacid, it furnished a green colour indicating the presence of steroids in the plantmaterials.
228 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
Test for Phenolic Compounds
The powdered plant material 1 g was boiled with distilled water andfiltered. When the filtrate was treated with neutral ferric chloride solution, itgave blue colouration, indicating the presence of phenolic groups.
Test for a-amino Acids
1 g of powdered fruits were boiled with water and filtered. An aqueousportion was drop onto a filter paper, allowed to dry and sprayed withninhydrin reagent. The filter paper was allowed to dry at room temperatureand then kept in an oven at 110°C for a few minutes. Pink spots wereobserved on the filter paper showing the presence of a-amino acid.
Test for carbohydrates
1 g of dried powdered fruit was boiled with distilled water and filtered.The filtrate was introduced into a test tube and a few drops of 10% a-naphtholwas added and shaken.
The test tube was then inclined at an angle of 45° and concentratedsulphuric acid was added slowly along the side of the test tube. A red ring wasformed between the two layers, showing the presence of carbohydrate in theplant material.
Test for Acidic or Basic Compounds
1 g of powdered plant materials was extracted with distilled water andfiltered. When the filtrate was treated with bromocresol green indicatorsolution, the resultant solution turned blue indicating the presence of organicbasic, when the resultant solution did not turn blue or red it is neutral.
Test for Tannins
1 g of powdered plant material was boiled with water and the filtratewas divided into two portions.
Jour. My an. Acad. Arts & Sa 2005 Vol. III. No. 4(ii) Botany 229
(a) A few drops of ferric chloride test solution was added to thefiltrate, a deep blue colour was produced indicating the presenceof tannins.
(b) A few drops of lead subacetate solution was added to the filtrate.Precipitates were formed, revealing the presence of tannins.
Thus, preliminary phytochemical tests have indicated the presence ofalkaloids, glycosides, reducing sugars, saponin glycosides, steroids, phenoliccompounds, a-amino acid, carbohydrates, organic base or neutral and tannins.The test has shown the absence of cyanogenic glycosides in the plantmaterials. The result was shown in table 1.
Table (1) Preliminary Phytcchemical Test on the fruits of genusMomordica L.
Type of product
1. Alkalodis
2. Reducing Sugars
3. Saponin
4. Cyanogenicglycosides
5. Glycosides
6. Steroids
7. Phenolic groups
8. a-amino acids
9. Carbohydrates
Test reagent
Mayer's reagent
Dragendorff s reagentSodium picrate solution
Benedict's solution
Distilled water
Cone: S/A & Sodiumpicrate paper
10% lead acetate
Jenzene, Aceticanhydride, and cone:S/A
FeCl3 solution
^inhydrin reagent
0%a-Naphthol,Conc:S/A
Observation
White ppt
Orange ppt
Yellow ppt
Brick-red ppt
Marked frothing
No colouration
White ppt
Green colour
Blue colour
*ink colour
Red ring
Plants
M,
+
-
+
+
+
+
+
M2
-
+
+
+
+
+
M3
+
-
+
+
+
+
+
M4
+
-
+
+
+
+
+
M5
- +
-
+
+
+
+
230 Jour. Myatu AcadL Arts & Sc 2005 Vol. III. No. 4(ii) Botany
Type of product
10. Organic acidic
or basic.
11. Tannin
Test reagent
Bromocresol green
(a) Ferric chloride testsolution
(b) lead subacetatesolution
Observation
blue -> Basic
yellow - •Acidic
No colourchange -> N
deep blue colour
Brown ppt
Plants
Mi
basic
.+
+
M2
N
+
M3
N
+
+
M4
N
.+
+
M j
basic
+
+
Mi
M2
M3
M4
M5
= M.charantinh.
= M.dioica Roxb.
= M.cochinchinensis Spreng.
= M.subangulata Blume
= M.muricata L.
+ = Present
- = absent
N = Neutral
Jour. Myan. Acad. Arts <ft 5c. 2005 Vol. III. No. 4(ii) Botany 231
Extraction and Isolation ofCharantin
Unripe fruit Powder (100 g)I Pet.ehter(b.p60-80C)
4, 600 ml
fat Marc(rejected) 80% EtOH 500 ml
EtOH Sol" M ^I KOH solution (pHIO)
basifiedSol" J
Ether
Ether jextractdistillation i
Crude CharantinI crystallization with EtOH {
CharantinUnripe fruit of Momordica were cut into small slices and dried in the
oven at the temperature of 50°C. The dried material were powdered by meansof pulverizer. 100 g of unripe fruit powder were mixed with 600 ml ofPet.ether (bp. 60"-80'C) and refluxed for (6) hours and then filtered. The marcwas repeated again for (6) hours with Petether and filtered. The marc wasmixed with 80% alcohol and extracted at reflux temperature for (6) hours andthen filtered. The filtrate was basified with potassium hydroxide solution tillpHIO and kept for (48) hours. The resulting solution was diluted with waterand extracted with diethyl ether. The diethyl ether portion was washed withwater, and then with dilute hydrochloric acid, and again with water.Anhydrous sodium sulphate was added to the diethyl ether portion and keptover night. The ether was filtered and concentrated to get residue (crudecharantin). The residue was dissolved in minimum amount of alcohol and keptin the refrigerator. The crystal was filtered, and the crystals was recrystallizedwith ethyl alcohol.
Charantin is a non-nitrogenous neutral substance, and the meltingpoint is 272°C.
The melting points of crude charantin and purified charantin weredetermined by means of capillary method. The result were shown in the table(2).
Table (2) Melting point of crude and purified charantin
Crystal
Crude charantin
Purified charantin
Observed
268-269°C
268-270°C
Reported
268-270°C
269-272°C
Thin Layer Charomatograpay
The purified Charantin crystal was applied on the silica gel G thinlayer chromatogram. The plate was developed in methanol and benzene,solvent systems at the ratio of 2:8. Then the plate was dried and the spot wasvisualized with iodine vapour. The Rf value of purified charantin was 0.5.
Ultraviolet absorption spectroscopy
The wave length of maximum absorption for the charantin compoundwas measured by using ultraviolet absorption spectrophotometer which X maxwas 206 nm.
Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 233
CO
i?nc
5 O
o Vi $
? £ £w CO (TJ
s z w
cmh -
?i¥ S§ o
o o
01 169
6EE9H
81-996
€6X921
f N N n N00 N- tO O i—
' N w n n N(O (B (O u'; 1
cr. CM « o<O C) <- N C9 fi 6 N fls a to in ico i- <• o 8>O! t - i - (M (N
<3>» CO
p
rem
I" CN
Xfl
(\ n Q9 <O
v £ t S B?E t «- 55 •••O 00 -r- r-
nCM
p o
»8<S> fO
5 =I™ {J
q . -c5
ifa; 5
00 00 C} 30
f"̂ U^ (O CO
CO O! t - T"
*n (P C7 f*̂<o «S- in in
CO N .
o en t -co so
CO COh- CO
CM
ssCO 0)
00 O
o
CO
JoCO
o 6 oCO
234 Jour. Myan. Aca<L Arts & Sc. 2005.Vol. III. No. 4(ii) Botany
Infrared spectroscopy
The infrared spectra of charantin showed the presence of conjugatedC=C band at 1639 cm"1, C-O stretching band at 1263, exocyclic methyl bandat 907 cm'1 and hydroxyl group band at 3435 cm"1. The infrared spectra ofcharantin compound was shown in Figure No 1.
FTIR Absorption Peaks and their Assignment of Compound Charantinis shown in table (3).
Table (3) FTIR Absorption Peaks and Their Assignment of CompoundCharantin
No. Wave Vibrational mode Functional groupnumber
1. 3435 UC-H of alcohol & acid
2.
3.
4.
5.
6.
7.
8.
9.
10.
3025
1639
827
2963
1456
1385
1364
1183
1104
UC-H of vinylidene
uc=c of alkene
5CH of vinylidene
UC-H of methyl and methylene
8CH of methyl and methylene
§CH of gemdimethyl group
uc-o of saturated secondaryalcohol
• \ vH• y"*i y~*i
• H
C H 3 - 1
CH2 - •
> / XCH3
• \ / H
Jour. My an. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 235
Discussion
Five species of the genus Momordica L. have been studied in thisresearch. Most of the species are wild grown and some species are cultivatedfor their edible fruits. The preliminary phytochemical examination was carriedout on the fruits of five species. The test indicated that all the fruits showedthe presence of alkaloid, glycosides, reducing sugar, saponin glycoside,steroids, phenolic compound, a-aminoacid, carbohydrates, organic base orneutral and tannin. The tests shown that the cyanogenic glycoside was absentfrom all fruits.
The active compound charantin was extracted from the fruits ofM.charantia L. The compound charantin showed activity on diabetes.According to the literature, the charantin is a mixture of equal parts of Psutisterol glucoside and 5, 25-stigmastadien.3|3-ol glucoside (A .A Olaniyi,1975).
The melting point of the isolated compound was identical with themelting point of charantin. The thin layer chromatogram showed a single spotso that the isolated compound was pure. The maximum absorption of isolatedcharantin compound was found at 206 nm. According to IR spectrum, theisolated compound showed all the functional group of charantin. So that theisolated compound was charantin.
Conclusion
The fruit and leaves of M.charantia L.(Bitter Melon) are used toreduce the levels of blood sugar. For centuries, bitter Melon has been used asfood and medicine in Asia, as therapeutic remedy in a variety of illnesses suchas leukaemia, diabetes, asthma, insect bites, maladies and stomach problems.
According to literature M.charantia L. fruit also show anti-HFVproperties. M.charantia L. fruits has shown the presence of charantincompound which can control diabetes (Lestile Tylor, 2002).
Furthermore investigation, some species of the genus Momordica L.fruits and leaves also show effect on diabetes and also have antimicrobialactivity.
236 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
ReferencesBacker, L.H. & Bakhuizen, A.F. (1963). Van Den Brink. Flora of Java.Vol. l.N.V.P.
Noordhoff Groningen, The Netherlands.
Burkill, L.H. (1935). A Dictionary of the Economic Products of Malay Penisula. Vol II.Grown agent for the Oxford University Press, London.
Harbone, J.B. (1989). Phytochemical Methods. Chapman and Hall, London.
Hooker, J.D. (1879). Flora of British India. Vol II. L. Reeve and Company England.
Hundley, H.G. (1978). List of Trees, Shrubs,Herbs and Principal Climbers, etc. 4th Rev.ed.Swe Daw Oo Press, Mayangon.
Hutchinson, J. (1967). The Genera of Flowering Plants. Vol II. The Clarendon Press, Oxford.
Kirtikar, K.R & Basu, B.D. 1938. Indian Medicinal Plants. Vol II 2nd ed. The Prabasi PressCalcutta.
Kirtikar, K.R. & Basu, B.D. (1938).' Indian Medicinal Plants. Vol II. 2nd ed. The PrabasiPress, Calcutta.
Roxburgh, W. (1832). Flora Indica (or) Description of Indian Plants. New Delhi, India.
Tylor, L. (2002). Herbal secrets of the Rainforest, 2nd Edition, Sage Press, Internet.
