PHYTOCHEMICAL SCREENING FOR KHELLIN IN SOME
KHELLA SEEDS (Ammi visnaga and Ammi majus) CULTIVATED
IN THE SUDAN.
By Esmahan Al Tyib Hassan Abosalah B.Sc. (Agric) Honours University of Khartoum 2000
A dissertation Submitted In Partial Fulfillment of the Requirements for the Degree of Master of Science in Food Science and Technology
Supervisor Dr. Khogali Elnur Ahmed Ishag
Department of Food Science and Technology Faculty of Agriculture University of Khartoum
December - 2004
1
DEDICATION
To the soul of my father and my brother To my mother, brothers, sisters Who were very helpful throughout the study To my dear friends and colleagues
With love and respect XáÅt{tÇ
2
Acknowledgements
Firstly I am most grateful to Allah for assistance, health and patience that gave to complete this work.
I am greatly indebted to my supervisor Dr. Khogali Elnur Ahmed
Ishag for his constructive and continuous guidance, friendly support and persistent encouragement.
I would also like to express my sincere thank and gratitude to Dr.
Hassan El Subki Khalid from the Medicinal and Aromatic Plant Institute, for his advise and interest.
I wish to express my great gratitude to the staff of Food Science and Technology Faculty of Agriculture, University of Khartoum, for their great assistance and help.
My gratitude is also extended to the staff of the Medicinal and aromatic plant research institute for their help and encouragement.
Also I am indebted to Mr. Arafat and Ms. Thouayba for their help during collection of plant material.
Sincere thanks are due to my brother and sister for their continuous help and moral support during my study.
Also my thanks are extended to all my friends and colleagues for their wonderful encouragement and help.
3 ABSTRACT Five samples of Ammi visnaga and Ammi majus seeds were collected from different sources in the Sudan. Phytochemical screening of extracts of A. visnaga and A. majus revealed the presence of various components such as saponin, Ttriterpens, Alkaloids, Flavoneids, Tannins and coumarins in addition to khellin. Extracts obtained from A. visnaga and A. majus seeds were distinguished by colour reaction where they gave faint yellowish green and dirty brownish green colour respectively. Extractions with chloroform and ethanol 60% for seeds of A. visnaga and A. majus samples were performed. Khellin was identified by thin layer chromatography. The identification was carried out by reaction colour, UV366 and Rf value were determined and khellin was identified by comparison against standards. Quantitative determination of khellin revealed variation in khellin content in the studied samples. It was found that A. visnaga collected from Shambat in 1993 and 1998 gave 0.627% and 1.892% khellin respectively, while A. majus seeds collected from Omdurman and Elobeid markets and from Shambat in 1993 did not contained khellin The study revealed that the extracts of all samples of A.visnaga under investigation contained variable amount of khellin and the highest content was found in A. Visnaga collected at Shambat in 1998.
4 ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ ﺧﻼﺻﺔ اﻷﻃﺮوﺣﺔ ﺟﻤﻌ ﺖ ﺧﻤ ﺴﺔ ﻋﻴﻨ ﺎت ﻣ ﻦ ﺑ ﺬور اﻟﺨﻠ ﺔ ﻣ ﻦ ﺛﻼﺛ ﺔ ﻣ ﺼﺎدر ﻣﺨﺘﻠﻔ ﺔ ه ﻲ ﺳ ﻮق أم درﻣ ﺎن
واﻷﺑﻴﺾ وﻣﺰرﻋﺔ ﺷﻤﺒﺎت. ﺗﻢ ﻣﺴﺢ آﻴﻤﻴﺎﺋﻲ ﻟﻤﺴﺘﺨﻠﺺ ﻧﺒﺎت ﺑﺬر اﻟﺨﻠ ﺔ اﻟﺒﻠﺪﻳ ﺔ ( A. visnaga)
واﻟ ﺸﻴﻄﺎﻧﻴﺔ (A. majus) ﻟﻤﻌﺮﻓ ﺔ ﺑﻌ ﺾ اﻟﻤﻜﻮﻧ ﺎت اﻟﻜﻴﻤﻴﺎﺋﻴ ﺔ وأوﺿ ﺤﺖ اﻟﺪراﺳ ﺔ وﺟ ﻮد أﻧ ﻮاع
ﻣﺨﺘﻠﻔ ﺔ ﻣ ﻦ اﻟﻤﺮآﺒ ﺎت ﻣﺜ ﻞ اﻟﺘﺎﻧﻴﻨ ﺎت ، اﻟﻔﻼﻓﻮﻧﻮﻳ ﺪات وأﺷ ﺒﺎﻩ اﻟﻘﻠﻮﻳ ﺎت اﻟ ﺼﺎﺑﻮﻧﻴﻦ ﺛﻼﺛ ﻲ ﺗ ﺮﺑﻴﻦ
واﻟﻜﻮﻣﺎرﻳﻦ ﺑﺎﻹﺿﺎﻓﺔ إﻟﻰ ﻣﺮآﺐ اﻟﺨﻠﻴﻦ.
ﻟﻘ ﺪ ﺗ ﻢ ﺗﻤﻴ ﺰ ﺑ ﻴﻦ ﺑ ﺬور ﺑ ﺬرة اﻟﺨﻠ ﺔ اﻟﺒﻠﺪﻳ ﺔ (A. visnaga) واﻟ ﺸﻴﻄﺎﻧﻴﺔ (A. majusِِ) وذﻟ ﻚ
ﺑﺘﻔ ﺎﻋﻼت ﻟﻮﻧﻴ ﺔ آ ﺸﻔﻴﺔ ﺣﻴ ﺚ أﻋﻄ ﻲ ﻣ ﺴﺘﺨﻠﺺ اﻟﺨﻠ ﺔ اﻟﺒﻠﺪﻳ ﺔ ﻟ ﻮن أﺻ ﻔﺮ ﻣﺨ ﻀﺮ ﺑﻴﻨﻤ ﺎ أﻋﻄ ﻲ
ﻣﺴﺘﺨﻠﺺ ﺑﺬور اﻟﺨﻠﺔ اﻟ ﺸﻴﻄﺎﻧﻴﺔ ﻟ ﻮن ﺑﻨ ﻲ ﻣﺨ ﻀﺮ آ ﺬﻟﻚ ﺗ ﻢ اﺳ ﺘﺨﻼص اﻟﺨﻠ ﻴﻦ (khellin) ﻣ ﻦ
اﻟﺒﺬور ﺑﺎﺳﺘﺨﺪام اﻟﻜﻠﻮروﻓ ﻮرم (Chloroform) واﻹﻳﺜ ﺎﻧﻮل ﺑﺘﺮآﻴ ﺰ Ethanol 60%) %60) ،
آﻤﺎ ﺗﻢ ﻓﺼﻞ اﻟﺨﻠﻴﻦ ﻋﻦ ﻃﺮﻳ ﻖ اﺳ ﺘﺨﺪام اﻟﻜﺮوﻣﻮﺗﻮﻏﺮاﻓﻴ ﺎ ذات اﻟﻄﺒﻘ ﺔ اﻟ ﺮﻗﻴﻘ ﺔ Thin-Layer)
(Chromatography أﻣﺎ اﻟﺘﻌﺮف ﻋﻠﻰ اﻟﺨﻠﻴﻦ ﻓﻘﺪ ﺗﻢ ﻋ ﻦ ﻃﺮﻳ ﻖ ﺗﻔﺎﻋ ﻞ ﻟ ﻮﻧﻲ آ ﺸﻔﻲ ﺑﺎﻹﺿ ﺎﻓﺔ
إﻟ ﻰ ﻣﻄﻴﺎﻓﻴ ﺔ إﻧﺒﻌ ﺎث ﺿ ﻮﺋﻲ ﻋﻨ ﺪ اﻷﺷ ﻌﺔ ﻓ ﻮق اﻟﺒﻨﻔ ﺴﺠﻴﺔ (UV366) وﻗﻴﻤ ﺔ Rf-value ﻣ ﻊ
ﻣﻘﺎرﻧﺘﻬﺎ ﻣﻊ ﻣﺮآﺐ ﻣﺮﺟﻌﻲ ﺧﻠﻴﻦ.
آﺬﻟﻚ ﺗﻢ ﺗﺤﺪﻳﺪ ﻧﺴﺒﺔ اﻟﻤﺎدة اﻟﻔﻌﺎﻟﺔ (اﻟﺨﻠﻴﻦ khellin) ﻓﻲ آ ﻞ اﻟﻌﻴﻨ ﺎت وﻗ ﺪ أﻇﻬ ﺮت ﻧﺘ ﺎﺋﺞ
اﻟﺘﺠﺮﺑﺔ أن هﻨﺎﻟﻚ ﻓﺮوق ﻣﻌﻨﻮﻳﺔ ﻓﻲ ﻧ ﺴﺒﺔ اﻟﺨﻠ ﻴﻦ ﺑ ﻴﻦ اﻟﻌﻴﻨ ﺎت اﻟﻤﺨﺘﻠﻔ ﺔ ﻟﻘ ﺪ وﺟ ﺪ أن ﻧ ﺴﺒﺔ اﻟ ﺨﻠ ﻴﻦ
ﻓ ﻲ ﺑ ﺬر اﻟﺨﻠ ﻪ اﻟﺒﻠﺪﻳ ﻪ A. visnaga اﻟﺘ ﻲ ﺟﻤﻌ ﺖ ﻣ ﻦ ﺷ ﻤﺒﺎت ﻓ ﻲ ﻋ ﺎم 1993 و 1998 آﺎﻧ ﺖ
آﺎﻵﺗﻲ 0.627 ، 1.892% ﻋﻠﻰ اﻟﺘﻮاﻟﻲ ، ﻓﻴﻤﺎ وﺟﺪ ان ﺑﺬر A. majus اﻟﺘﻲ ﺟﻤﻌﺖ ﻣ ﻦ ﺷ ﻤﺒﺎت
1993 وﻣﻦ ﺳﻮق أم درﻣﺎن واﻷﺑﻴﺾ ﺟﻤﻴﻌﻬﺎ ﻻ ﺗﺤﺘﻮي ﻋﻠﻲ ﺧﻠﻴﻦ.
وﻗ ﺪ ﺧﻠ ﺼﺖ اﻟﺪراﺳ ﺔ إﻟ ﻰ أن آ ﻞ ﻋﻴﻨ ﺎت ﺑ ﺬ ر ﻧﺒ ﺎت اﻟﺨﻠ ﺔ اﻟﺒﻠﺪﻳ ﺔ A. visnaga
ﺗﺤﺘﻮي ﻋﻠﻰ اﻟﺨﻠﻴﻦ (khellin) ﺑﻨﺴﺐ ﻣﺨﺘﻠﻔ ﺔ وأن أﻋﻠ ﻲ ﻧ ﺴﺒﺔ وﺟ ﺪت ﻓ ﻲ ﺑ ﺬ ر اﻟﺨﻠ ﺔ اﻟﺒﻠﺪﻳ ﺔ
(A. visnaga) اﻟﺘﻲ ﺗﻢ ﺟﻤﻌﻬﺎ ﻣﻦ ﺷﻤﺒﺎت ﻋﺎم 1998 .
5 LIST OF CONTENTS Page
Dedication……………………………………………………………………………………………... i
Acknowledgement …………………………………………….………………………………... ii
Abstract ………………………………………………………..……………………………………... iii
Arabic Abstract …………………………………………………………………………………... iv
List of Contents ………………………………..………………………………………………... v
List of Tables……………………………………..………………………………………………... viii
List of Figures ………………………………..…………………………………………………... ix
List of Plates ………..…………………………..…………………………………………………... x
CHAPTER ONE: INTRODUCTION……………………………………………... 1 CHAPTER TWO: LITERATURE
REVIEW……………………………… 3
2.1. Classification of khella plant……………………………………………………... 3
2.2 Geographical distribution of Khella……………………………………………... 3
2.3 Description of Khella……………………………………………………………………... 4
2.3.1 Botanical Description of Khella………………………………………………... 4
2.3.2 Microscopical description of Khella……..…………………………………... 5
2.4. Cultivation details…………………………………..……………………………………... 5
2.5 Chemical composition of khella…………………………………………………... 6
2.6. Medicinal usage of Khella…………………………………………………………... 7
2.6.1. Therapeutic action of Khella……………………………………………………... 8
2.6.2. Dosage of Khella………………………………………………………………………... 9
6 2.6.3. Contraindication of khella………………………………………………………... 9
2.6.4. Toxicity of Khella…………………………………………………………...... 10
2.7. Other usage of Khella………………….………………………………………………... 10
2.8. Chemistry of ingredients……………………………………………………………… 10
2.9. Extraction of khella…………………………………….………………………………... 11
2.10. Determination of active Ingredients…………………………………………... 14
CHAPTER THREE: MATERIALS AND METHODS……..………… 17
3.1 Materials…………………………………………………………………..……………………... 17
3.2 Chemicals and reagents………………………………………………………………... 17
3.3. Preparation of the Sample…………………………………………………………... 18
3.4 Preliminary phytochemical Screening of Khella………………………... 18
3.4.1 Preparation of extract………………………………………………………………... 19
3.4.2. Qualitative test for Unsaturated sterols and triterpenes…………… 19
3.4.3. Qualitative test for Alkaloids…………………………………………………... 19
3.4.4. Qualitative test for Flavonoids……………………………………………... 20
3.4.5. Qualitative test for tannins………………………………………………………... 20
3.4.6. Qualitative test for saponins…………………………………………………... 21
3.4.7. Qualitative test for cyanogenis glycoside………………………………... 21
3.4.8. Qualitative test for Anthraquinone glycoside…………………………... 21
3.4.9. Qualitative test for coumarins……………………………………………………….. 22
3.5. Preliminary examination for identification of Ammi visnaga and Ammi majus seeds………………………………………………………………………... 22
3.6. Methods for examination of extract compounds………………………... 22
7
3.6.1. Extraction according to the methods adopted by Stahl and
Schild (1981) ………………………………………………………………………………………... 22
3.6.2. Extraction according to the methods adopted by Wayne -
Ivie(1978) ………………………………………………..……………….…………………………... 23
3.6.3. Detection of the Khella's components……….……………………………... 23
3.7. Moisture content………………………….………………………………………………... 25
3.7.1. Determination of Khellin…………………………………………………………... 25
3.7.2. γ – pyrone determination as Khellin……………………………………….... 25
CHAPTER FOUR: RESULTS AND DISCUSSIONS………… 29 4.1 Preliminary phytochemical screening of extract from Ammi visnage and Ammi majus…………………………………………………………………... 29
4.2 Identity examination……………………………………………………………………... 31 4.3 Thin-layer chromatlography of extracts of A. visnaga and A. majus seeds………………………………….………………………………………………………... 33 4.4. Variation of khellin content in A. visnaga and A. majus seeds from different sources……………………..…………………………………………………... 44
CHAPTER FIVE: SUMMARY AND RECOMMENDATION 50
REFERENCES…………………………………………………………………………………... 52
8 LIST OF TABLES
Table Title No. 1. Preliminary phytochemical screening of extract from A.
visnaga and A. majus…………………………………………………………... 30
2. Colour reaction seeds extracts…………………………………………... 32 3. TCL for chloroform extract from A. visnaga and A. majus
seeds in S.I………………………………………………………….……………… 34
4. TLC for chloroform extracts of A. visnaga and A. majus
in S.II………………………………………………….…………………………… 38
5. TLC for chloroform extracts of A. visnaga and A. majus in
S.III……………………………………………………………………………………… 41 6. TLC data of 60% ethanol extract of A. visnaga and A.
majus seeds in S.IIV………………………….………………………………… 45 7. Means of khellin content of A. visnaga and A. majus from
different sources…………………………………………..……………………… 48
9 LIST OF FIGURES
Fig. Title No.
1. Structure of xanthotoxin, Bergapten and isopimpinellin… 12
2. Structure of Khellin, visngin and Khellol glycoside………… 13
3. Means of khellin content of A. visnaga and A. majus
from different sources ……………………………..……….………………… 49
10 LIST OF PLATES
Fig. Title No.
1. Plant of Ammi visnaga……………………………………………………...… 28
2. Plant of Ammi majus…………………………………………………………… 29
3. Solvent system Chloroform : ethyl acetate ……………………… 36
4. Solvent system Hexane : ethyl acetate : methanol…………… 40
5. Solvent system Benzene : ether………………………………………… 43
6. Solvent system ethyle acetate……………………..……………………… 47
11 CHAPTER ONE
INTRODUCTION
The plants which contain active medicinal chemical constituents and give a definite physiological response in the treatments of diseases in humans or other animals are called medicinal plants, (Hamayun, 2003).
The close relationship between man and his environment continues even today as large proportion of people in the developing countries still live in rural areas. Furthermore, modern pharmacopoeia still contain at least 25% drugs derived from plant and many other which are synthetic analogues built on prototype compounds isolated from plants, (De-Silva, 1997).
Recently medicinal plants are receiving more attention to treat and cure, since these plants are easily obtained, less costy and have less side effects as synthetic. Some countries like India, China, Egypt and Greece are famous for using medicinal plants (Auterhoff, 1953).
The production of medicinal and aromatic plants is assuming an important economic role in such countries as India and China due to the increasing demands to meet local consumption as well as export,
(ElBushra, 1999). In Sudan people have accumulated a lot of knowledge about the use of different plants to treat many diseases traditionally.
Khella is a medicinal plant which is obtained from two species of
Ammi for example, Ammi majus has been used medicinally in the Middle
East for centuries in the treatment of leukoderma, However, it was established that the photosensitizing and pigment stimulating a gents in
12 this plant were a group of compounds known as linear furanocoumarins or psoralens, (Wayne-Ivie, 1978).
Ammi visnaga is an indigenous herb on the waste lands in the Nile
Delta it holds different common names in different countries. It is called
Khella in some Arab countries, tooth pick herb or Bishop's weed in
England, Chile and Europe (Hussian, 1981; ELFiky et al, 1989). The chromone Khellin of visnaga fruit a long with visnagin and Khellol glycoside, is a potent coronary vasodilator and bronchodilator and is used for the treatment of coronary insufficiency, angina pectoris and bronchial asthma (Mustafa et al., 1984). In addition, preparation of visnaga in Egyptian folk medicine is marketed for treatment of renal colic and renal stones (Mesbah, 1992). Although khella is well known in
Sudan, it is production is limited to areas in Northern state as winter crop. A survey carried out revealed that growers are unaware of the proper cultural practices (ElBushra, 1999).The objectives of this work are :-
1. To distinguish between A. majus and A. visnaga seeds relying
on a chemical reaction of their extracts.
2. To extract khellin from the seeds using different solvents.
3. Screening the main chemical components of khella seed extracts.
4. To vary different solvent systems for chromatographic separation
of khellin.
5. Quantitative determination of khellin in different khella seeds
extracts.
13 CHAPTER TWO
LITERATURE REVIEW
2.1. Classification of khella plant: (Kartesz, 2004)
Ammi visnaga and Ammi majus
Division: Flowering plant
Class: Dicoty ledons
Sub class: Rosidae
Order: Apiales
Family: apiaceae (umbelliferae)
Genus: Ammi
Species: Ammi visnaga, Ammi majus
Common name: Ammi, Khilla, Khella, khillal, Pick tooth, tooth pick, Bishops weed Visnaga and Khella Sheitaani (Batanoumy, 2002).
2.2 Geographical distribution of Khella:
Visnaga origin is Egypt and Mediterranean, it grows in Syria,
Saudi Arabia, Iraq, Sudan, Canary Islands and southern France (Hussian,
1981 and Saad et al., 1988). Ammi majus groups are in coastal regions of the Southern United states (Dollahite et al., 1978). Also it grows widely in Egypt as medicinal plant (Hussain, 1981 and Mesbah, 1992).
14 2.3 Description of Khella:
2.3.1 Botanical Description of Khella:
Visnaga and majus are common annual herbaceous plants about one metre high. The stem is erect, solid, cylindrical, finally longitudinally straited and has more smooth branches (Collett, 1952;
Hussian, 1981; Abuzeid 1986; Saad et al, 1988; ElGhazali, 1997 and
Lonchamp, 2000).
The leaves are numerous finely divided linear segments with capillary vessels. The flowers are white in umbel with numerous rays (up to 150) and tight with indented petals, in volucre with one, two or several pinnatisect bracts with filiform strap. fruits are ovate oblong (2-2.5 mm) distinguished from. Ammi majus because of slightly protruding sides
(Lonchamp, 2000).Moreover, Flowers appears in March to April. the fruits odor is slightly aromatic (Treas and Evan, 1989), it appears in
May to June and it is somewhat pear-shape, (2.5-2.6) mm length, brown in color and slightly bitter in taste (Collett, 1952). Also it is divided in two fruits each has one seed (Hussian, 1981 and Saad et al, 1988). At maturity the umbellets come together and the umbel appears nearly closed (Abuzied, 1986).
15 2.3.2 Microscopical description of Khella:
Epidermis of pericarp consists of polygonal cells, elongated on the ridges with occasional crystals of calcium Oxalate and finely straited cuticle on hair. Mesocarp, formed of parenchyma, traversed longitudinally by schizogenous viltae, each surrounded by large, slightly radiation cells and traversed in the ridges by vascular bundles each forming a crescent around a comparatively large vita and accompanied by fibers and reticulate lignified cells, the inner larger of the mesocarp consists of large, polygonal, brown-walled cells with thick porous inner wall. Endocarp, composed of narrow tangentially elongated cells, some of these being regularly arranged in groups, variously oriented. Adhering to brown seed-coat which is formed of similar but wider and some what shorter cells. Endosperm, consists of polygonal, thick-walled, cellulosic parenchyma, contain fixed oil and numerous small oval aleuronic grains, each enclosing a minute rounded globoid and micro-rosetta crystal of calcium oxalate with a dark center. Copophore, traversed by a vascular strand of fibers and spiral vassels (Batanoumy, 2002).
2.4. Cultivation details:
Khella grows in all types of soil, but light and loam is desirable
(Abuzeid, 1986).Bown (1995) reported that the plant prefer a well- drained soil in sunny position, succeeding in ordinary garden soil, it
16 tolerates a pH in the range of (8.3 to 8.8) and it is resistant to drought and salinity.
Ammi seeds are sown directly during winter with high relative humidity in October to mid December with seeds rate 2-3 kg/faddan. and the irrigation water applied six time during the growing season (Hussian,
1981 and Abuzeid, 1986). ElBushra, (1999) revealed that seeds yield component were positively effected by sowing date, Hence the optimum time for sowing visnaga in Khartoum state is from fifteenth of
November to the December. After ripening, plants are tied in bundles and staked in heaps for a few days to dry then threshed on clean floor, the stalks are separated by hand and the fruit collected (Hussian, 1981)
Moreover, (Balbaa et al., 1972) reported that the best time for collection of Ammi majus fruits is in the stage when they become mature, but still unripe to achieve the highest yield of furanocoumarins.
2.5 Chemical composition of khella:
Ammi majus fruits contain coumarins and coumarin glycoside and it yields not less than 0.5% of Ammoidin (xanthotoxin), 0.3% Ammidin
(imperatorin) and 0.01% of majudin (bergapaten).
Furanocoumarins have also been produced by cell suspension culture (Botanoumy, 2002). Other furanocoumarins were indentified and
17 isolated from the fruit such as bergapten, isopimpinellen, ammajin, marmesin and majurin (Balbaa et al, 1973).
The Ammi visnaga fruits contents furanochromones at least 1.0%
γ- pyrone derivatives (Stahl and Schild, 1981). Moreover it is included khellin was about (0.3-1.2%) visnagin about (0.05-0.3%), khellol, khellenin, khellinol, ammiol, visammiol, khellinone and visnaginone
(Botanoumy, 2002).
Treas and Evans (1989) stated that visnagin content of seed was about 0.1 percent and khellol glycoside 0.3 percent, also the fruits were content of visnadine, it marketed as antinginal agents (Durarte et al.,
1997; Vickycrouse et al, 1998). In addition to that, the seeds contents of pyrancoumarins (visnagans) was about (0.2-0.5%) comprising visnadin, samidin, dihydrosamidin and trace of xanthotoxin and ammidin; also they have flavones derivative, volatiles and fixed and protein substances
(Botanoumy, 2002).
2.6. Medicinal usage of Khella:
Ammi visnaga, Ammi majus and other photosensitizing plant are cultivated even today in some parts of world as sources of these medicinally important compounds (Wayne-Ivie, 1978). Ammi majus has been used medicinally in Middle East countries for treatment of leukoderma, skin depigmentation and vitiligo (Pathak et al., 1974).
18 The furanochromones, khellin and visnagin were detected in the primary rib channels and endosperm of ripe achenes have been used for centuries in Egyptian folk medicine for treatment of kidney and bladder stones, and in western medicine against Asthma (Franchi et al., 1987;
Bown, 1995; Tilgner, 1999 and Batonoumy, 2002). Moreover Khellin and Visnagin have received considerable attention, because of their vasodilatory properties and are responsible for their acute hypotensive effects (Chen, 1993 and Durarte et al., 2000). It used to treat angina pectoris, as antispasmodic and in the treatment as cardiovascular diseases (Rauwald et al., 1994).
Recently small amount of volatile oil has been used in treatment of skin diseases like vitiligo and psoriasis (Tilgner, 1999). Bown (1995) reported that, the seeds have a strongly antispasmodic effects on the smaller bronchial muscles they also dilate the bronchial, urinary and blood vessels with out affecting blood pressure.
2.6.1. Therapeutic action of Khella:
Therapeutic action of Khella seeds were such as antispasmodic agents with specific action as a coronary vasodilator and bronchodilator, calculi in reanel colic, and it decreases the toxicity of the cardiac glycoside digitoxin due to the coronary vasodilator. Antiarrhythmic effects and improves blood supply to myocardium (Vickycrouse et al.,
1998). In addition to this visnaga was found to the reduce the pain
19 caused by trapped kindney stone and helps ease the stone down into the bladder, by relaxing the muscles of urethra (Chevallier, 1996).
Visnagine drug inhibited vascular smooth muscle contractility probably by acting at multiple sites to decrease the availability of Ca+2 required for activation (Ubeda et al, 1991; Durate et al., 1997).
2.6.2. Dosage of Khella:
According to Treas and Evan (1989) who stated that khellin is the most important active constituent, it has been synthesized and commercially available in the form of tablet and injections. Tilgner
(1999) reported that the one teaspoon crushed seeds per cup of water, in fused for 25 minutes, or 1: 3 dry liquid extract, 20 – 60 drops in a little water for 1 – 4 times per day, it was bitter and slightly aromatic, also the preparation of soaked visnaga seeds has been used as diuretic drink
(ElFiky et al., 1989), the plant has no side effects (Chevallier, 1996).
2.6.3. Contraindication of khella:
Khella decreases the toxicity of cardic glycoside digitoxin due to the coronary vasodilator and antiarrhythomic effect. It was contraindicated in pregnancy due to the emmenagogue and uterine stimulating activity of Khellin. Moreover it may cause photodermatites to sensitive individuals (Vickycrouse et al., 1998 and Tilgner, 1999).
20 2.6.4. Toxicity of Khella:
Abuzied (1980) reported that the Ammi genus include different species, which are morphological and chemically extremely different.
Both species are considered as the most noxious weeds to sugar-beat in
Mediterranean countries e.g. Ammi majus in Italy and Ammi visnaga in
Spain (Villarias, 1992). On the other hand, the daily oral administration of the seeds of Ammi visnaga at dosages 0.25 and 5g/kg are toxic to goats and they profuse salivation and bouts of diarrhea. Also the renal lesions showed a good correlation with increased levels of urea and decrease on concentration of total protein, calcium and Inorganic phosphate in serum as well as the histopathological result pointed to damage to kidney and liver(Ahmed,1989).
2.7. Other usage of Khella:
The fruiting pedicel is used as a tooth pick (Usher, 1974) and whilst the seeds have been used as a tooth cleaner (Chevallier, 1996).
Growing visnaga a long the borders of sweet pepper (Capsicum annuum) fields had favorable effect, because of its association with beneficial insects (Bugg and Wilson, 1990).
2.8. Chemistry of ingredients:
The yields of the psoralen obtained from the Ammi majus seeds extracts such as xanthotoxin, Bergapten, Isopimpinellin and isoimperatorin were crystallized from ether (Wayne-Ivie, 1978).
21 Visnaga seeds owed their medicinal and economic importance to the furanochromone khellin. According to Collett (1952) and Treas and
Evans (1989) it is a 2-methyl 5, 8 dimethoxy 6, 7 furanochromone, it is crystalline and it occurs to extent of about 1% white needle also has a bitter taste odorless, melts at 153–155ºC. It is soluble in chloroform
,alcohol and less soluble in ether and it is sparingly soluble in petroleum ether and cold water, but more soluble in boiling water (Balabaa,1981)
Khellin is accompanied by other crystalline compounds visnagin and khellol glycoside. Visnagin is a small needle like crystal of melting point
144ºC, it is available as by product in the isolation of khellin. Khellol glycoside is soluble in ether and occurs as needle crystals of melting point 175ºC (Collett, 1952; Hussain, 1981 and Treas and Evans, 1989).
2.9. Extraction of khella:
Extraction stages are in the sequence, petroleum ether, chloroform, ether, acetone and methanol. They are carried out in either a percolation tube or a soxhlet apparatus. The chloroform extract contains many furanocoumarins (Stahl, 1969).
Strakowsky and Badran (1958) isolated the furanocoumarin by extracting the fruit, with ethanol. Marmesin, the aglycone- of ammajin, was isolated by Abu Mustafa et al., (1958) from the fruits by extracting it first with benzene to remove other furanocoumarins then with alcohol.
22 Fig. (1): Structure of Xanthotoxin, Bergapten and Isopimpinellin
1. Xanthotoxin (8 – methoxy psoralen)
CH3
O
O O O
2. Bergapten (5 – methoxy psoralen)
O O O
O
CH3
3. Isopimpinellin (5, 8 – dimethoxy psoralen). CH 3 O O O O
O
CH3 23 Fig. (2): Structure of Khellin, Visngin and Khellol glycoside
1. Khellin
CH3
O O | |
O CH3 O O
CH3
2. Visnagin O CH3O | |
O CH O 3 H
3. Khellol glycoside
O CH3O | |
O O CH2O.C6H11O5
24 2.10. Determination of active Ingredients:
Several methods have been published for estimation of furanocoumarins, the gravimetric method was the first method used for estimation of furanocoumarins present in the fruits of Ammi majus and it is still beeing used (Akacic and Kustrak, 1958,1961). The colorimetric method has been adopted for the estimation of total furanocoumarins
(Blazek and Stary, 1965). The spectrophotometric method has been used for the estimation of individual furanocoumarins, mainly xanthotoxin, imperatorin and bergapten after their separation either by paper chromatography which was adopted in most cases (Bseyrich, 1966,
1967) or with Thin–layer Chromatography (Karawy et al., 1970). Both the spectrophotometric and colorimetric methods gave nearly similar results (Balbaa et al, 1973).
Thin–layer Chromatography–spectrophotometric method depend on separating furanocoumarins by thin- layer chromatography, followed by their elution and spectrophotometric determination (Balbaa et al.,
1972).
The method was successfully applied to analysis of khellin in raw materials and in pharamaceutical.It is based on quantitative thin – layer chromatography with the spectrodensitometric evaluation of the spots obtained, which determined khellin in respective concentration ranges
25 0.02–0.14 mg/ml (Bebawy et al., 1998). In addition to this the production of furanochromones, khellin and visnagin in the embryos were determined by a normal phase high-performance liquid chromatography method, it produced 0.57% khellin and 0.72% visnagin on dry weight. During vegetative growth of the plant,contains relatively low levels of furanocohromones but high concentration were observed at early flowering (ElFiky et al., 1989).
Also Franchi et al. (1985) studied the presence of furanochromones khellin and visnagin in the various organs of Ammi visnaga at different development stages.The determination was carried out using high-performance liquid chromatography. They found that unripe fruits had the highest content of both chromones yielding 43% khellin and 0.68% visnagin. Mesbah (1992) determinated khellin and visnagin in Ammi visnaga fruits and in renal teas by high-performance liquid chromatography. They were extracted with chloroform from samples of powdered evaporated to dryness and the residue dissolved in
67% methanol. Aliquots using 67% methanol as an isocratic eluent and ultraviolet detector at 254nm.
According to Ynazgrka et al. (1998) who stated that a new, simple and rapid solid Phase extraction method for the determination of furanochromones and pyranocoumarins in Ammi visnaga L. fruits and
26 pharmaceutical by reversed–phase high–performance liquid chromato- graphy was developed, the isolation of compounds examined was carried out on Octadecyl Baker Bond SPE columns using various concentration of methanol, acetonitrile and tetrahydrofuran in water. High and reproducible recoveries were obtained. In addition to this a new, simple and rapid capillary electrophoresis method was developed for the identification and quantitative determination of two medically active constituents. Khellin and visnagin in the extracts of Ammi visnaga fruits were extracted with methanol and the extract were directly injected without any purification and pre-separation processes. Coumarin was used as internal standard for quantitation and the limits of detection for
Khellin and visnagin were 2.36 and 1.97mg/ml, respectively using UV detection at 245nm, (Gunaydin and Erim, 2002).
27 CHAPTER THREE
MATERIALS AND METHODS
3.1 Materials:
Ammi visnaga and Ammi majus (L.) Lamarck (Apiaceae) seeds were collected from three different sources. Samples 'A' and 'B' of Ammi majus were collected from Omdurman and Elobeid markets respectively,
Samples 'C' of ammi visnaga and 'D' of ammi majus were collected from the farm of the faculty of Agriculture university of Khartoum at
Shambat in season 1993 .Sample 'E' of Ammi visnaga in season 1998 and
Sample 'F' were Tablets obtained from the company of Abdelmoneim
Medical Industries ,Sudan.
The seed were identified at Medicinal and Aromatic Plants
Research institute of the National Research Council (NRC)Herbarium segment were deposited at NAPRI .
3.2 Chemicals and reagents:
The chemicals used were as follows:
• Chloroform.
• Ferric chloride.
• Conc. sulphuric acid .
• Sodium sulfate anhydrous.
• Methanol.
28 • Ethylacetate.
• Acetonitrile.
• Hexane.
• Petroleum ether.
• Benzene.
• Acetic anhydride.
• Mayer's reagent.
• Aluminum hydroxide.
• Gelatin salt reagent.
• Hydrogen peroxide.
• Ammonium hydroxide.
3.3. Preparation of the Sample:
Whole seeds of A . visnaga and A. majus were milled in a mortar then the powdered samples are kept in glass containers.
Praeparatio of extract:
3.3. Preliminary phytochemical Screening of Khella:
Phytochemical screening of Ammi seeds was done according to the method adopted by Harborne (1973).
29 3.4. 1 Preparation of extract:
Ten grams of the powder A.visnaga and A.majus seeds were refluxed with 100 ml of 80% ethanol for four hours .After cooling the solution was filtered and enough 80% ethanol was passed through the residue volume of the fitrate and was filled to 100 ml.
This prepared extract was used for the various tests.
3.4.2 Qualitative test for Unsaturated sterols and triterpenes:
Ten mls of extract was evaporated to dryness on a water path and the cooled residue was stirred several times with petroleum ether to remove most of the coloring material. The residue was then extracted with 20ml chloroform. the chloroform solution was dehydrated over anhydrous sodium sulphate. Five millitres portion of the chloroform extract was mixed with 0.5ml of acetic anhydride followed by 2 drops of conc sulphuric acid. The gradual appearance of green, blue pink to purple colour was taken as an evidence of the presence of sterols (green to blue )and triterpenses (Pink to purple) in the sample.
3.4.3 Qualitative test for Alkaloids:
seven and ahalf millitres of extract was evaporated to dryness on a water bath. Five millitres of 2NHCl was added and stirred while heating on the water bath for 10 minutes, cooled filtered and few drops of
Mayer's reagent were added A slight turbidity or heavy precipitate in tube was taken as presumptive evidence for the presence of alkaloids.
30 3.4.4 Qualitative test for Flavonoids:
seventeen millitres of extract was evaporated to dryness on water bath, cooled and the residue was defatted by several extractions with petroleum ether and the defatted residue was dissolved in 30ml of 80% ethanol and filtered.
The filtrate was used for the following tests:
• Three millitres of the filtrate were taken in a test tube then,
1ml of 1% aluminum chloride solution in methanol was added.
• Three millitres of the filtrate were taken in a test tube and 1ml
of 1% potassium hydroxide solution was added. Formation of
yellow colour indicated the presence of flavonoids.
3.4.5 Qualitative test for Tannins:
seven millitres of extract was evaporated to dryness on water bath.
The residue was extracted several times with n-hexane and filtered. The insoluble residue was stirred with 10ml of hot saline solution. The mixture was cooled, filtered and the volume of the filtrate was adjusted to 10 ml with more saline solution.five millitres of this solution was treated with few drops of gelatin salt reagent and few drops of ferric chloride were added to another portion of this solution. The formation of blue black or green was taken as an evidence for presence of tannins.
31 3.4.6 Qualitative test for Saponins:
One gram of the powdered seeds was placed in a clean test tube.
10ml of distilled water was added and the tube was stoppered and vigorously shaken for 30 seconds. The tube was then allowed to stand and observed for the formation of honeycomb, which persisted for least an hour, was taken as evidence for presence of saponins.
3.4.7 Qualitative test for Cyanogenic glycoside:
Three grams of powdered seeds were placed in Erlenmeyer flask and sufficient water was added to moisten the sample followed by 1ml of chloroform. A piece of freshly prepared sodium picrate paper was carefully inserted between a split crock which was used to stopper the flask,a change in colour of the sodium picrate paper from yellow to various shades of red was taken as an indication of presence of cyanogenic glycoside.
3.4.8 Qualitative test for Anthraquinone glycoside:
One gram of the powdered seeds were boiled with ten millitres of
0.5N KOH containing one millitre of 3% hydrogen peroxide solution.
The mixture was extracted by shaking with 10ml of benzene. Then 5ml of benzene extract was shaken with three millitres of 10% ammonium hydroxide solution, and two layers were allowed to separate. The presence of anthraquinones was found to have assumed pink or red colour.
32 3.4.9 Qualitative test for Coumarins:
Three grams of powdered seeds were boiled with 20ml distilled water in a flask and a filter paper attached to the flask was to be saturated with the vapour then a spot of 0.5N KOH was put on it then the filter paper was inspected under UV light. The presence of coumarin was indicated if the spots have found to be adsorbed the UV light.
3.5. Preliminary examination for identification of Ammi visnaga and
Ammi majus seeds:
According to the procedure of Stahl and Schild (1981) 0.5 gram powdered seeds were placed in a clean test tube. Four millitres methanol were added and the tube was shaken .The extract was filtered. Then five drops of conc H2SO4 were added for colourification, formation of the dirty brownish green colour indicated for presence of furanocoumarin and faint yellow green colour indicated for presence of furanochromones.
3.6. Methods for examination of extract compounds:
3.6.1. Extraction according to the methods adopted by Stahl and
Schild (1981):
0.5grams powdered seed were refluxed with 10ml ethanol 60%
(v/v) for 30 min. the cool solution was filtered and the filtrate was concentrated on water bath to 5ml.
33 3.6.2. Extraction according to the methods adopted by Wayne –Ivie
(1978):
Hundred grams of powdered seeds were placed in 500ml flask and extracted with 250ml of chloroform on shaking for 24 hours. The slurry was filtered under vacuum and the residue was extracted three more times with chloroform. The chloroform extracts were dried over anhydrous sodium sulfate then the chloroform removed by vacuum distillation in rotary evaporator the residue was partitioned between acetonitrile and hexane 100ml each to remove the lipids and the hexane phase was discarded the acetonitrile was adjusted to 100ml.
3.6.3. Detection of the Khella's components:
The prepared extracts were used to detect the components of
Khella by using Thin-layer chromatography .
The chromatography was done according to the methods of Stahl
(1969).
Preparation of the plate:
Thirty grams of silica gel GF 254 were shaken for 2 minutes with
60ml distilled water in a 250ml stoppered conical flask The slurry was spreaded using spreader making 0.25mm thick layer of 5 glass plate 20
X 20cm the coated plate were allowed to dry at room temperature then activated at 105º for one hour hot plates were stored and allowed to cool
34 down in desiccator over blue gel. The stationary phases was: Silica gel type GF254:
Application of sample: the test sample to be separated was applied by means of capillary tubes.
o Mobile phase:
S.I. Chloroform: ethylacetate 60 : 30.
S.II. Hexane: Ethylacetate: Methanol 25 : 25
S.III. Benzene: ether 60 : 3.
S.IV Ethylacetate.
Preparation of locating reagents:
The following locating reagents were used:
• HCl conc.
• Anisaldehyde reagent.
0.5ml Anisaldehyde was mixed with ten millitres acetic Acid .followed by one millitre methanol andfive milllitres conc sulphuric acid.
Visulisation:
The chromatograms were initially examined under the long wave of ultraviolet lamp (366nm), after being freed of solvent. The chromatograms were then sprayed with suitable reagent the spots attain maximum, color intensity the plate was sprayed with Anisaldehyde sulphuric acid reagent and ferric chloride HCl (conc).
35 The Rf values of developed compounds was calculated as follow:
Distance moved by spot for a component Distance moved by solvent front 3.7. Moisture content:
The moisture content was determinated according to Stahl and
Schild (1981). Two grams of powdered seeds were placed in preheated crucibles, and dried in an oven at 105ºC for two hours, then the crucibles were transferred to desiccator to cool and reweighed the moisture content was calculated using the following equation:
W − W Moisture content (%) = 2 1 ×100 W0
W0 = sample weight.
W1 = weight of (sample +crucible) before drying
W2 = weight of (sample +crucble) after drying.
3.7.1. Determination of Khellin:
γ-pyrone was determinated as khellin content according to the procedure of Stahl and Schild (1981). As follows:
3.7.2. γ – pyrone determination as Khellin:
0.25gm of dried powdered seeds were placed in 100ml round bottom flask. 50ml distilled water were added. Then the mixture was heated under reflux condenser for 30 minutes. The hot mixture was filtered by filter paper and the residue was washed twice with 5ml of hot
36 distilled water for each The collected aqueous filtrate was transferred to
250 separating funnel, allowed to cool down to room temperature. 40ml of chloroform were added and then shaken four times then, the layer allowed to separate. The chloroform extract was dried over anhydrous, sodium sulphate, then filtered under vacuum and evaporated by Rotary evaporator to dryness. The residue was dissolved on warming with conc.
HCl : H2O mixture of equal volumes; 30ml solvent were taken three time. Then the solution allowed to cool drown to room temperature. The solution was placed in a 100ml volumetric flask and completed to total volume with the HCl : H2O solvent mixture, the solution was filtered with glass filter funnel. The absorbent of resulted solution was measured at 400nm, against the HCl : H2O mixture as blank solution.
The following formula was used for calculating the percentage of
γ-pyrone expressed as Khellin was taken as the value of (1% cm) at maximum of 400nm
10000.E Khellin (%) = 1% E1cm .(100 − P)b E = absorption of extract.
1% E1cm = 112. P = moisture content of seeds a percentage. b = volume of seeds in (g).
37
Plate 1. Plant of Ammi visnaga
38 Plate 2. Plant of Ammi majus
39 CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Preliminary phytochemical screening of extract from Ammi visnage and Ammi majus
Table (1) shows that the two species contain Saponins, Triterpens,
Alkaliods, Flavonoids, Tannins and Coumarins where as unsaturated sterols, cyanogenic glycoside and Anthraquinones glycoside were not detected.
These findings are comparable to the results of Fields,
(2003),who mentioned that A.visnaga contain furanchoromones such as khellin and visnagin, coumarins, Flavonoids, Voltile oils and phytosterol. Also Babaa et al (1972) found furanocoumarins in the mature unripe fruits of A. majus .Simillarly Stahl and Schild (1981) reported that A. visnaga fruits contained γ–pyron coumarins and flavon derivative .
40 Table (1) Preliminary phytochemical screening of extract from A. visnaga and A. majus.
Compounds A. visnaga A. majus Reaction Foam Saponins + + (3-4-6) Pink colour Triterpenes + + (3-4-2) No reaction Unsterated sterols _ _ (3-4-2) Slight turbidity Alkaloids + + (3-4-3) Yellow colour Flavonoids + + (3-4-4) Cynogunic No reaction _ _ glycoside (3-4-7) Precipitate and Tannins + + green colour (3-4-5) Uv light absorbed Coumarins + + (3-4-9) Anthraquinone No reaction _ _ glycoside (3-4-8)
+ presence – absence
41 4.2 Identity examination:
The colour reaction of extract obtained from A. visnaga and A. majus seeds collected from Omdurman market(sample A) gave faint green colour, while the extracts of A. majus seeds collected from
Shambat in 1993 (sample D) gave dirty brownish green colour indicating presence of furanocoumarins and confirming, the identify of A. majus was reported by Stahl and Schild (1981). Extracts of A. visnaga seeds collected from Shambat in season 1993 (C) and season 1998 (E) gave faint yellowish green colour indicating presence of furanochromones.
These results agree with the finding of Stahl and Schild (1981). who reported that furanochromones which are found only in A. visnaga form yellowish green colour. On treatment with concentrate sulphuric acid due to formation of pyrylium salt where as furanocoumarins of A. majus gave dirty brownish green colour. Reaction colour differences for extracts of seeds of same variety may be due to differences of sources and locality of collection which can be influenced by climatic factors on the components, mainly temperature and light as well as stage of harvest and storage conditions.
42
Table (2) : Colour reaction of seeds extracts :
Extract Reaction Compounds
A Faint green Furanocoumarins
B Dark green Furanocoumarins
C Faint yellowish green Furanochromones
D Dirty brownish green Furanocoumarins
E Faint yellowish green Furanochromones
Where :
A: Extract of A. majus seeds collected from Omdurman market
B: Extract of A. majus seeds collected form Elobeid market
C: Extract of A. visnaga seeds collected from Shambat (1993)
D: Extract of A. majus seeds collected from Shambat (1993)
E: Extract of A. visnaga seeds collected from Shambat (1998)
43 4.3 Thin-layer chromatlography of extracts of A. visnaga and A. majus seeds:
The following solvent systems were found to give acceptable separation for khellin:
The solvent system S.1(Cholorform : Ethylactate 60 :30) gave fairly good separation for component of the extracts. It can be inferred from table (3) and plate No (3) that samples A, B and D gave two spots on TLC plates developed in S.1which observed under UV366 one of the spots appeared blue while the other gave yellow fluorescence. The Rf– values for the compounds in that solvent system were found to be (0.83,
0.85) for sample A, (0.85, 0.91) for sample B and (0.79, 0.85) for sample
D. The spot which gave blue fluorescence under UV366 did not give colour reaction on treatment with the spray reagent and remained unidentified as khellin. However the other spots of samples C and E with
Rf–values 0.85 and 0.83 respectively which exhibited yellow colour fluorescence under UV366 gave yellow colour with the spray reagent were identified by comparison against standard (F) and tablets (G) as khellin.
44 Table (3) TLC for chloroform extract from A. visnaga and A. majus seeds in S.I*
Colour reaction sample Spot No Rf value UV366 Spray reagent 1 0.83 Blue - A 2 0.85 yellow - 1 0.83 Blue - B 2 0.85 yellow - 1 0.85 Yellow Yellow C 2 0.88 Blue - 1 0.79 Blue - D 2 0.85 Yellow - 1 0.83 Yellow Yellow E 2 0.89 Blue - F 1 0.86 Yellow Yellow G 1 0.86 Yellow Yellow
*S.I:- chloroform : ethylctate ( 60 : 30) where :
A: Extract of A. majus seeds collected from Omdurman market
B: Extract of A. majus seeds collected from Elobied market
C: Extract of A. visnaga seeds collected from Shambat (1993)
D: Extract of A. majus seeds collected from Shambat (1993)
E: Extract of A. visnaga seeds collected from Shambat (1998)
F: Standard khellin
G: Tablet of khellin from Abdelmoniem Medical Industries,
Sudan
45
Plate No. (3). TLC of extracts in Solvent system Chloroform : ethyl acetate (60 : 30)
1. A. majus (Omdurman) 2. A. majus (Elobied)
3. A. visnaga (Shambat, 1993) 4. Tablet
5. A. majus (Shambat, 1993) 6. A. visnaga (Shambat, 1998)
7. Standard khellin
46 indicating presence of khelin in A. visnaga seed extract. It was noticed that samples A, B, D showed the same Rf values of 0.85 with yellow fluorescence but had no reaction colour with spray reagent indicating absence of khellin in A. majus.
Samples C and E showed the same number of spots, having almost similar Rf–values. It can be inferred from table (3). that the components with Rf value 0.85 and 0.83 which appeared as yellow spot under
UV366 and was also detected when the plate was sprayed with anisaldehyde reagent were identified as khellin, while the other components of Rf value 0.88 and 0.89 which appeared as blue spots under UV366 but were not detected on spraying with the reagent were not khellin. The yellow spots noticed in all the sample had almost same
RF values of the reference sample F of khellin. The fluorescence of these spots under UV366 were the same but with varying degree of intensity indicating that the extract of all samples under investigation contained khellin. Plate No (4) and table (4) demonstrate the separation of the same samples as in plat No (1) excepted that the solvent system S.II (Hexane:
Ethylactate : Methanol 25 : 25 : 5) was used instead. The spots were found to be more than those of solvent S.I, indicating that solvent S.II was better resolving than solvent S.I. This solvent system revealed three components in samples A, B and D of A. majus and six components in
47 samples C and E of A. visnaga. furthermore table (4) shows that the standard khellin in sample F and G had Rf value 0.92 and appeared as yellow fluorescent under UV366 and developed yellow colour when treated with anisaldehyde-sulphuric acid spray reagent. The spots which have Rf value (0.215, 0.51) for sample A, (0.18, 0.46) for sample B and
(0.17, 0.41) for sample D, exhibit blue colour under UV366 and they were not detected with the spray reagent. Samples C and E had yellow spots at Rf value 0.89 and 0.92 respectively and both were identified in reaction to reference compound of F and G, indicating clearly that C and
E contained khellin appeared as yellow fluorescence spots under UV366, however,such yellow florescent spots with related Rf in sample A, B and
D did not appear when the plate was sprayed with anisaldehyde- sulpheric acid, indicating that the three samples did not contain khellin.
48 Table (4) TLC for chloroform extracts of A. visnaga and A. majus in
S.II*
colour reaction sample Spot No Rf value Spray UV366 reagent 1 0.215 Blue - A 2 0.51 Blue - 3 0.90 Yellow - 1 0.18 Blue - B 2 0.46 Blue - 3 0.88 Yellow - 1 0.11 Yellow - 2 0.18 Blue - 3 0.31 yellow - C 4 0.44 Blue - 5 0.59 Blue - 6 0.89 Yellow Yellow 1 0.17 Blue - D 2 0.41 Blue - 3 0.88 Yellow - 1 0.10 Yellow - 2 0.17 Blue - 3 0.29 yellow - E 4 .44 Blue - 5 0.59 Blue - 6 0.92 Yellow Yellow F 1 0.92 Yellow Yellow G 1 0.91 Yellow Yellow
*S.II : Hexane : Ethylactyate : Methanol (25 : 25 : 5)
49
Plate No. (4). TLC of extracts in Solvent system Hexane : ethyl acetate : methanol (25 : 25 : 5)
1. A. majus (Omdurman) 2. A. majus (Elobied)
3. A. visnaga (Shambat, 1993) 4. Tablet
5. A. majus (Shambat, 1993) 6. A. visnaga (Shambat, 1998)
7. Standard khellin
50 Plate No (5) and table (5) demonstrate the separation of the chloroform extracts from A. visnaga and A. majus seeds in the solvent system SIII which was (benzene : ether) ( 60 : 3).
The separated components were found to be less shifted indicating that SIII was less polar than S.I and S.II. This solvent system revealed other major compounds in sample A, B and D which gave seven spots and the examination of the developed plate under UV366 showed the presence of fluorescent compounds. The separated components appeared as blue and yellow spots. It is clear from table (5) that samples C and E have shown four spots with the almost same Rf values.
The components in samples C and E with Rf values o.089 and
0.095 respectively appeared as yellow spots under UV366 and when the plate was spray with anisaldehyde-sulphuric acid reagent was identified by comparison on TLC against standard khellin as refrence samples F and G, indicating that the extracts of two samples of A.visnaga under investigation contained khellin. The solvent system revealed other compounds which gave blue colour under UV366 and did not appear when the plate was sprayed with anisaldehyde-sulphuric acid reagent.
These compounds remained unidentified .
51 Table (5) TLC for chloroform extracts of A. visnaga and A. majus in S.III* Colour reaction sample Spots No Rf values Spraying UV366 reagent 1 0.035 Blue _ 2 0.083 Yellow _ 3 0.113 Blue _ A 4 0.209 Blue _ 5 0.443 Yellow _ 6 0.562 Yellow _ 7 0.598 Blue 1 0.041 Blue __ 2 0.071 Yellow _ 3 0.101 Blue _ B 4 0.209 Blue _ 5 0.449 Yellow _ 6 0.562 Yellow _ 7 0.598 Blue 1 0.089 Yellow Yellow 2 0.143 Blue _ C 3 0.476 Yellow _ 4 0.598 Blue _ 1 0.041 Blue _ 2 0.083 Yellow _ 3 0.137 Blue _ D 4 0.239 Blue _ 5 0.479 Yellow _ 6 0.589 Yellow _ 7 0.658 Blue _ 1 0.095 Yellow Yellow 2 0.125 Blue _ E 3 0.419 Yellow _ 4 0.606 Blue _ F 1 0.089 Yellow Yellow G 1 0.089 Yellow Yellow
*S.III : Benzene : Ether (60 : 3)
52
Plate No. (5). TLC of extracts in Solvent system Benzene : Ether (60 : 3)
1. A. majus (Omdurman) 2. A. majus (Elobied)
3. A. visnaga (Shambat, 1993) 4. Tablet
5. A. majus (Shambat, 1993) 6. A. visnaga (Shambat, 1998)
7. Standard khellin
53 Plate No (6), demonstrates that the solvent system Ethylactate gave fairly good separation for the components of extract from A. visnaga and A. majus seed extracted by 60% ethanol, the data is shown in table (6).
The samples of A. majus gave similar components which might vary in their concentration. However, the samples of A. visnaga showed some variation for the different components indicated by colour reaction in UV366 and Rf values.
Examination of the developed plate under UV366 showed the presence of fluorescent compounds. The separated components appeared as blue and yellow spots. Samples A, B and D showed similar spots, having same Rf values. The spots which exhibited blue colour under
UV366 were not detected on spraying with the locating reagent.
Samples C and E have shown four spots having the same RF values. The components with Rf value 0.823 appeared as yellow spots under UV366 and also when the plate was sprayed with Ferric chloride.
Qualitatively, the yellow spots which were noticed in samples C and E showed the same Rf value of the reference samples F and G, which have RF value 0.823and appeared as yellow colour under UV366 and when the plate was sprayed with Conc. HCl, this indicates that the compound of yellow colour of the samples is most propaby khellin.
However, in the sample A, B and D this compound did not appear when the plate was sprayed with Ferric chloride.
54 Spraying with anisaldehyde reagent revealed a greater intensity of colour than that obtained with HCl reagent, also the colour reaction with
HCl was unstable. It can be inferred from the Rf value and colour under
UV366 that all the tested samples with both extracting solvents contain khellin compound with varying concentrations.
4.4. Variation of khellin content in A. visnaga and A. majus seeds from different sources:
Table (7) and Fig (3) show that the highest content of khellin was observed in extract of A. visnaga seeds collected from Shambat in 1998 with 1.892% which is significantly high (P < 0.05). The lowest content was observed in extract of A. majus seeds collected from Omdurman ,
ElObeid market and Shambat in 1993
A. visnaga seeds collected from Shambat in 1993 and in 1998 had
1.892% and 0.627% khellin respectively. These results are comparable to Poal et al., (1964) results who found 1.17% khellin in A. visnaga;
Wagner et al., 1984 found a range of 0.3 – 1% khellin content in A. visnaga and Sahl and Schild (1981) found a range of 1.5–2% khellin content in A. visnaga.
55 Table (6) TLC data of 60% ethanol extract of A. visnaga and A. majus seeds in S.IV* Colour reaction sample Spots No Rf value Spray UV366 reagent 1 0.176 Blue _ A 2 0.26 Blue _ 3 0.823 Yellow _ 1 0.176 Blue _ B 2 0.26 Blue _ 3 0.823 Yellow _ 1 0.082 Yellow _ 2 0.176 Blue _ C 3 0.26 Blue _ 4 0.823 Yellow Yellow 1 0.176 Blue _ D 2 0.2635 Blue _ 3 0.825 Yellow _ 1 0.088 Yellow _ 2 0.235 Blue _ E 3 0.294 Blue _ 4 0.823 Yellow Yellow F 1 0.823 Yellow Yellow G 1 0.823 Yellow Yellow
* S.IV: ethyl acetate Where : A: Extract of A. majus seeds collected from Omdurman market B: Extract of A. majus seeds collected from Elobied market C: Extract of A. visnaga seeds collected from Shambat (1993) D: Extract of A. majus seeds collected from Shambat (1993) E: Extract of A. visnaga seeds collected from Shambat (1998) F: Standared khellin G: Tablet of khellin from Abdelmoniem Medical Industries, Sudan
56
Plate No. (6). TLC of extract in Solvent system Ethyl acetate
1. A. majus (Omdurman) 2. A. majus (Elobied)
3. A. visnaga (Shambat, 1993) 4. Tablet
5. A. majus (Shambat, 1993) 6. A. visnaga (Shambat, 1998)
7. Standard khellin
57 Generally, khellin content in A. visnaga was significantly at (P <
0.05) higher level as compared to that of the A. majus. The result is inconformity with Stahl and Schild (1981) results who found khellin in
A. visnaga, where as khellin was not observed in A. majus. Variation in khellin content between A. visnaga and A. majus could be attributed to genetic variation between the two species. Variation in khellin content within the A. visnaga samples may be due to the effect of season 0f collection, period of storage and climatic conditions.
58
Table (7). Means of khellin content of A. visnaga and A. majus from different sources
Sample Khellin content (%)
A 0
B 0
C 0.627b
D 0
E 1.892a
LSD 0.10
coefficient of variance CV% 4.14
Where:
A: Extract of A. majus seeds collected from Omdurman market B: Extract of A. majus seeds collected from Elobied market C: Extract of A. visnaga seeds collected from Shambat (1993) D: Extract of A. majus seeds collected from Shambat (1993) E: Extract of A. visnaga seeds collected from Shambat (1998)
59 2 1.8 1.6 1.4 1.2 1 0.8 0.6 Khellin content (%) (%) content Khellin 0.4 0.2 0 ABCDE Samples
A A. majus C A. visnaga
Fig. 3. Means of khellin content of A. visnaga and A. majus from different sources
60 CHAPTER FIVE
SUMMARY AND RECOMMENDATIONS
The phytochemical screening of extracts of the seeds of Ammi plants has shown that both species of Ammi majus and Ammi. visnaga contain Triterpens, Alkaloids, Flavonoids, Tannins,
Saponions and Coumarins.
Unsaturated sterols, Cyanogenic glycoside and Anthraquinone glycoside were not detected. The two species could be distinguished by a sample test for their extracts A. visnaga gave faint yellowish green colour due to furanochromones where as A. majus gave dirty brownish green colour due to furanocoumarins.
Thin-layer chromatography (TLC) examination for khellin revealed that samples of A. visnaga probably contain khellin at varying concentrations which was clearly identified in A. visnaga extracts.
Acceptable result separation for khellin by (TLC) was obtained using the solvents systems:
Chloroform: Ethylacetate 60 : 30, Hexane : Ethylacetate : Methanol
25 : 25 : 5 and Benzene : Ether 60 : 3. Anisaldehyde sulphuric acid reagent was found to be a reliable locating reagent for khellin.
61 Khellin content in seeds was found to be affected by season of storage period and conditions on khellin stability need further study.
Recommendation:
1. A. visnaga seeds are a good source for khellin.
2. Further systematic study is required to find out the optimum
stage of harvest with the highest khellin content.
3. The affect of storage periods and climatic conditions on Khellin
stability need further study.
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