Cassia Senna L.) Compared to Samples from Erkweit, Red Sea State, Sudan

Nutritional, Phytochemical and Toxicity Characteristics of Local Sana (Cassia senna L.) Compared to Samples from Erkweit, Red Sea State, Sudan

Ayda Ali Khalifa Mohammed B.Sc. (Hon.) in Agronomy, Faculty of Agriculture, University of Zalingei, 1997

A Dissertation

Submitted to the University of Gezira in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science

Biosciences and Biotechnology (Biotechnology) Center of Biosciences and Biotechnology Faculty of Engineering and Technology

March 2015

Nutritional, Phytochemical and Toxicity Characteristics of Local Sana (Cassia senna L.) Compared to Samples from Erkweit, Red Sea State, Sudan

Ayda Ali Khalifa Mohammed

Supervision Committee:

Name Position Signature

Dr. Mutaman Ali Kehail Supervisor ……………..

Prof. Elnour Elamin Abdelrahman Co-Supervisor ……………..

Date: March, 2015

Nutritional, Phytochemical and Toxicity Characteristics of Local Sana (Cassia senna L.) Compared to Samples from Erkweit, Red Sea State, Sudan

Ayda Ali Khalifa Mohammed

Examination Committee:

Name Position Signature

Dr. Mutaman Ali Kehail Chair Person ……………..

Prof. Elnaeim Abd Allah Ali External Examiner ……………..

Prof. Ali Osman Ali Internal Examiner ……………..

Date: 25, March, 2015

Dedication

To my Family

Nutritional, Phytochemical and Toxicity Characteristics of Local Sana (Cassia senna L.) Compared to Samples from Erkweit, Red Sea State, Sudan Ayda Ali Khalifa Mohammed

Abstract

Cassia senna plant is well known as sanamakka. It grown in many areas in the Gezira State and in other States. Many nutritional and pharmaceutical uses of this plant were reported worldwide. The comparative nutritional composition, qualitative phytochemical screening, thin layer chromatography and toxicity, for Wad Medani (W) and Erkweit (E) samples of C. senna seeds (S) and leaves (L) were the aims of this study. The study was conducted during September 2014. The collected samples were prepared for the study experiments in the University of Gezira, Faculty of Engineering and Technology. Larvae of Anopheles and Culex mosquitoes were also collected for the toxicity test. The conclusions of this study revealed that, the highest nutritional contents in all samples was the carbohydrates (61.75%) followed by protein (17.93%), then ash (7.19%), fat (0.31%) and lastly fibers (0.09%), but Anova proved no significant differences between the four samples in their nutritional contents. The phytochemical screening, showed that, the leaves of Erkweit sample, contained glycosides, flavonoids, flavonone and alkaloids, as same as Erkweit seeds, which contained also sterols, while the leaves sample of Wad Medani resemble completely Erkweit seeds, but Wad Medani seeds sample, lack sterols and glycosides. The thin layer chromatography detected a total of three spots from the leaves and four spots from the seeds of Wad Medani samples, but the case was not similar for Erkweit samples, leaves part separated four spots (two from the polar and two from the apolar extracts), while that, five spots were separated from the seed part (two spots from the apolar extract and three from polar extract). The study recommended to get benefit of the nutritional contents from C. senna, irrespective of their source, also the pharmaceutical importance of such plant should be studied.

الخصائص التغذوية, الكيميائية النباتية والسامة لنبات السنمكة المحلي مقارنة بعينات من أركويت,

والية البحر األحمر, السودان

عايدة علي خليفة محمد

ملخص الدراسة

يعرف نبات "كاشيا سنا" بالسنمكة. ينمو في عدة مناطق في والية الجزيرة وفي الواليات األخري. غني

بالمجتمعات الحيوانية المائية كنتاج للشبكة الكثيفة من قنوات مشروع الجزيرة. العديد من اإلستخدامات التغذوية

والصيدالنية تم نشرها علي مستوي العالم. مقارنة المحتوي الغذائي, المسح الكيميائي النباتي الكيفي,

كروماتوغرافيا الطبقة الرقيقة والسمية لعينات بذور وأوراقالسنمكة من ود مدني )والية الجزيرة( وأركويت )والية

البحر األحمر( هي أهداف هذا البحث. تمت هذه الدراسة في الفترة سبتمبر 2014. تم إعداد العينات المجموعة

إلختبارات الدراسة في جامعة الجزيرة, كلية الهندسة والتكنولوجيا. تم أيضاً جمع يرقات بعوض االنوفلس

والكيولكس إلختبار السمية. خلص البحث إلي أن أعلي مكون غذائي لكل العينات هو الكربوهيدرات

)61.75%( يتبعه البروتين )17.93%(, الرماد )7.19%(, الدهن )0.31%( وأخي اًر األلياف )%0.09(,

ولكن إختبار تحليل التباين أوضح أن ال فرق معنوي بين العينات األربع في محتواها الغذائي. المسح الكيميائي

النباتي أوضح أن أوراق عينة أركويت إحتوت علي الجاليوكوسيدات, الفالفونويدات, الفالفونونات والقلويدات,

مثل بذور عينات أركويت, والتي تحتوي أيضاً علي اإلستروالت, بينما أو ارق عينة ود مدني تشبه كلياً بذور

أركويت, ولكن بذور عينة ود مدني تفتقر لإلستروالت والجاليكوسيدات. الدراسة حدد إختبار كروماتوغرافيا الطبقة

الرقيقة مجمل ثالثة بقع من األوراق وأربعة بقع من بذور عينات ود مدني, ولكن الحالة ليست متشابهة لعينات

أركويت, حيث فصلت جزء األوراق أربعة بقع )إثنان من المستخلص القطبي واثنان من المستخلص الالقطبي(,

بينما تم فصل خمسة بقع من البذور )إثنان من المستخلص الالقطبي وثالثة من المستخلص القطبي(. أوصي

البحث باإلستفادة من المكونات الغذائية لنبات السنمكة بغض النظر عن مصدرها, وكذلك بدراسة األهمية

الصيدالنية لهذا النبات.

List of Contents

Subject Page Dedication iii Acknowledgements iv Abstract v Arabic Abstract vi List of Contents vii List of Tables ix CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: LITERATURE REVIEW 3 2.1. Cassia senna 3 2.1.1 Scientific classification 3 2.1.2.Description 3 2.1.3 Uses 4 2.1.4 Chemical composition 5 2.2. Phytochemical analysis 7 2.2.1 Procedure 7 2.3. Thin layer chromatography 9 CHAPTER THREE: MATERIALS AND METHODS 3.1 Materials 10 3.2. Proximate analysis tests 10 3.2.1. Ash content 10 3.2.2. Oil content 10 3.2.3. Protein content 11 3.2.4. Crude fiber content 11 3.2.5. Carbohydrates content 12 3.3. Phytochemical screening tests 9

3.3.1 Test for the presence of alkaloids 12 3.3.2 Test for the presence of flavonoids and flavonones 12 3.3.3 Test for the presence of glycosides 12 3.3.4 Test for the presence of saponins 12 3.3.5 Test for the presence of steroids 12 3.3.6 Test for the presence of tannins 13 3.4. Thin layer chromatography test 13 3.5. Toxicity test of C. senna leaves and seeds powder 13 3.4. Statistical analysis 13 CHAPTER FOUR: RESULTS AND DISCUSSION 4.1 Proximate analysis of C. senna of Wad Medani and Erkweit areas 14 4.2. Qualitative phytochemical screening of C. senna 16 4.3. Thin layer chromatography test for C. senna 18 4.4. Toxicity of C. senna leaves and seeds powder against mosquito larvae 20 CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions 23 5.2. Recommendations 23 References 24

List of Tables

Table Title Page No

Profiles of the phytochemical screening, minerals and nutritional 2.1 6 contents of Cassia senna leaves and seeds

2.2 Qualitative phytochemical analysis by using different solvents 8

the proximate analysis of C. senna brought from Wad Medani and from 4.1 15 Erkweit areas

4.2 Qualitative phytochemical screening of C. senna leaves and seeds 17

Thin layer chromatography test for C. senna leaves and seeds brought 4.3 19 from Wad Medani area and Erkweit area

Toxicity of 0.6 g (per 250 ml) powder of C. senna leaves and seeds

4.4 (from Wad Medani and Erkweit areas) toward Anopheles and Culex 21

larvae

Chapter One Introduction

The name Senna is from Arabic sana. Cassia is an angiosperme widely distributed in Africa, in Latin America, and in Australia and Fiji. Firstly classified in Caesalpiniacae family, then in those of Leguminoseae, Cassia is now classified among the Fabaceae. This plant is a shrub which has a medium-size, 10-12 m tall, occasionally reaching 20 m. The bark is grey and smooth, and later with longitudinal fissures. The leaves are alternate, 15-30 cm long, compound, with 6-14 leaflets each ending in a tiny bristle. The flowers are bright yellow, in large, up to 60 cm long, upright, with pyramid-shaped panicles. The fruits are flat with indehiscent pod, 5-30 cm long, and constricted between the seeds. There are about 20 seeds per pod. The seeds are bean-shaped, greenish-brown, and 8-15 mm long (Gutteridge, 1997). The leaves, stems, roots, flowers and seeds of Cassia regardless the subspecies have been used for the treatment of several illnesses including mostly malaria, a tropical endemic disease with high morbimortality (Oyedunmade and Olabiyi, 2006). The preparation process of remedies was not clearly described and the dosages prescribed were approximate. Moreover, the treatments are supposed to be continued until recovery. According to the ethnic differences of populations from localities, the plant is used alone or in combination with other plants or with natural substances for the preparation, especially in decoction. For the treatment, people mostly used the preparations by oral administration route (Ejobi et al., 2007). The leaves are the most used parts’ the plant especially by African and Asian population in preparation of the herbal remedies. In Burkina Faso, fresh and dried leaves decoction is drunk with lemon juice or for body bath throughout the day to treat malaria and liver disorders (Nadembega et al., 2011). In Côte d’Ivoire, the decoction of leaves is administered orally (0.5 L, twice daily) for treating cough, stomach pains and malaria. Also, in Sierra Leone and Togo, the leaves decoction is drunk against malaria (Mbatchi et al., 2006) and used as antimicrobial (Souza et al., 1995). In Nigeria, the dried leaves are mixed with lemon’s leaves (Cymbopogon citratus), pawpaw’s leaves (Carica papaya), and the lime’s leaves (Citrus lemonum) and are boiled within an hour. The "tea" of the mixture is drunk against malaria (Ogunkunle and Ladejobi, 2006).

In Uganda, the leaves are picked, cleaned and chewed, and liquid swallowed to treat abdominal pains (Kamatenesi et al, 2011). In India, the leaves are cleaned thoroughly and boiled. The decoction is filted in which is added honey. This preparation is drunk against anemia and fever (Sati et al., 2010). In Laos, fresh and dried leaves taken as a vegetable which has sedative and euphorising effects (Bhagya and Sridha, 2009; Grisanapan, 2010). In Thailand, dried leaves are sprayed to be regularly consumed in capsule form as vegetable for its laxative effect and as sleeping pill (Kiepe, 1995; Ngamrojanavanich et al., 2006; Tapsoba and Deschamps, 2006). Other authors reported that, Cassia leaves decoction is drunk against constipation and hypertension and are inhaled in toothache (Thongsaard et al., 1996). The seeds are used to charm away intestinal worms and as antidote for snake and scorpion bites. The decoction of the mixture of Cassia and Ficus thonnigii fruits’ is drunk to prevent convulsions in children and to treat typhoid fever. In Sri Lanka and Thailand, the flowers and young fruits are regularly consumed as vegetable and for treating curries. It provides laxative and sleeping-aid effect (Kiepe, 1995). This dish is also anxiolitic and effective against dysuria (Ngamrojanavanich et al.,2006). C. siamea seems less toxic justifying its wide use in folklore medicine (Teangpook et al., 2011). Indeed, its stem bark’s aqueous extract showed less sub- chronic toxicity in male wistar rats (Mohammed et al., 2012). This extract and root’s aqueous extracts were found to be relatively not toxic on blood, hepatic and renal cells in wistar rats (Otimenyin et al., 2010).

The objectives of this study By using of Wad Medani and Erkweit samples of Cassia senna seeds and leaves as research materials, the aims can be stated as: 1- To determine the percentage of nutritional composition, 2- To run a qualitative phytochemical screening, 3- To determine the active ingredients through thin layer chromatography test 4- To test the toxicity using Anopheles and Culex larvae as a biomarkers.

Chapter Two Literature Review

2.1. Cassia senna Senna (from Arabic sanā), the sennas, is a large genus of flowering plants in the legume family Fabaceae, and the subfamily Caesalpinioideae. This diverse genus is native throughout the tropics, with a small number of species in temperate regions. The number of species is estimated to be from about 260 (Marazzi, 2006) to 350 (Randell and Barlow, 1998). About 50 species of Senna are known in cultivation (Huxley, 1992). 2.1.1 Scientific classification

Kingdom: Plantae (unranked): Angiosperms (unranked): Eudicots (unranked): Rosids Order: Fabales Family: Fabaceae Subfamily: Caesalpinioideae Tribe: Cassieae Subtribe: Cassiinae, (Huxley, 1992). 2.1.2. Description Senna includes herbs, shrubs, and trees. The leaves are pinnate with opposite paired leaflets. The inflorescences are racemes at the ends of branches or emerging from the leaf axils. The flower has five sepals and five usually yellow petals. There are ten straight stamens. The stamens may be different sizes, and some are staminodes. The fruit is a legume pod containing several seeds. Chamaecrista, Cassia, and Senna form a monophyletic group which some authors have called Cassia sensu lato. In 1982, the group was named Cassiinae and classified as a subtribe of the tribe Cassieae (Irwin and Barneby, 1982). The tribe Cassieae contains 21 genera and is now known to be polyphyletic, but the classification is still accepted because a revision of Fabaceae has yet to be published

(Lewis, 2005). The genus Senna has had a complex taxonomic history (Singh, 2001). What is now known as Senna was included by Linnaeus in his concept of Cassia in Species Plantarum in 1753 (Linnaeus, 1753). Philip Miller segregated Senna from Cassia in 1754 in the fourth edition of The Gardeners Dictionary (Miller, 1754). Until 1982, many authors, following Linnaeus, did not recognize Senna and Chamaecrista, but included them in a broadly circumscribed Cassia sensu lato. Phylogenetic analyses of DNA have shown that Chamaecrista, Cassia, and Senna are all monophyletic, but the relationships between these three genera have not been resolved. They are therefore shown in phylogenetic trees as a tritomy (Marazzi, 2006). 2.1.3 Uses Some Senna species are used as ornamental plants in landscaping. The species are adapted to many climate types. Cassia gum, an extract of the seeds of Chinese senna (S. obtusifolia), is used as a thickening agent. The leaves and flowers of Siamese cassia (S. siamea) are used in some Southeast Asian cuisines, such as Thai and Lao cuisines. They are known as khi-lek in Thai, and are used in curries (Teangpook et al., 2011). Senna italica ssp. italica (syn. Cassia obovata), often called neutral henna, is used as a hair treatment. It has effects similar to henna, but without the red color, giving hair more of a yellow tinge. The active component is an anthraquinone derivative called chrysophanic acid or chrysophanol (1,8-Dihydroxy-3- methylanthraquinone), which is also found in higher concentrations in rhubarb root. Chrysophanol has been reported to have antimicrobial (García-Sosa, 2006) and anti- inflammatory properties. It is also a component of the pheromone of the beetle Galeruca tanaceti (Kim, 2010). Sennas have for millennia played a major role in herbalism and folk medicine. Alexandrian senna (S. alexandrina) has long been traded commercially. Senna glycosides, or sennosides, are used in modern medicine as laxatives (Spiller, 2003). Senna drugs contain the dried leaves of S. alexandrina. The glycosides increase gastric fluid secretion and bowel motility, producing laxative action. Senna preparations are available in powders, granules, tablets, oral infusions, and syrups. It is also available in combination with the dietary fiber psyllium to add bulk to the bowel contents (Agarwal and Bajpai, 2010). The products are only recommended for

13 short-term use, and chronic use and abuse of senna has been associated with organ failure (Vanderperren, 2005). Utilized to treat conditions like constipation, hemorrhoids, reduce fevers, biliousness, bad breath, colic, gallstones, gout, jaundice, menstruation, mouth sores, obesity, boils, pimples, rheumatism, treat skin diseases and kill parasites. Experiments with senna leaf preparations are not available. A senna extract showed mutagenic toxicity in vitro. The pure substance, sennoside A, B, showed no mutagenic toxicity in vitro. An in vivo study with a defined extract of senna pod revealed no mutagenicity. The study was conducted over 104 weeks. Rats received up to 25 mg/kg body weight and showed no substance-dependent increase of tumors (MDidea.com, 2014). 2.1.4 Chemical composition Senna consists of the dried leaflets or fruits of Cassia senna (C. acutifolia) known in commerce as Alexandrian senna and of Cassia angustifolia commonly known as Tinnevelly senna. The senna plants are small shrubs of Leguminosae cultivated either in Somalia, the Arabian Peninsula or near the Nile River. T. senna is obtained from cultivated plants mainly in South India and Pakistan. Owing to the careful way in which the plant is harvested, the leaflets of the drug are usually little broken. Damaged leaves and lower quality products are often used for making galenicals. The senna pods (fruits) are collected during the same period as the leaves, then dried and separated into various qualities. The active principle of Senna was first isolated and characterized in 1941. The first two glycosides were identified and attributed to the anthraquinone family. These were found to be dimeric products of aloe emodin and/or rhein which were named sennoside A and sennoside B. They both hydrolyze to give the aglycones sennidin A and B and two molecules of glucose. Later work confirmed these findings and further demonstrated the presence of sennosides C and D. Small quantities of monomeric glycosides and free anthraquinones seem to be present as well. The active constituents of the pods are similar to those of the leaves but present in larger quantities. Two naphthalene glycosides isolated from senna leaves and pods are 6-hydroxymusicin glucoside and tinnevellin glucoside. Both compounds can be utilized to distinguish between the Alexandrian senna and the India senna, since tinnevellin glucoside is only found in the latter and the first only in the C. senna (Franz, 1993). Chemical, mineral and phytochemical profile was presented in Table (2.1).

Table (2.1) Profiles of the phytochemical screening, minerals and nutritional contents of Cassia senna leaves and seeds Leaves Seeds phytochemical Alkaloids Alkaloids Anthraquinones Anthraquinones Steroids Steroids Triterpenoids - Flavonoids - Glycosides - Minerals Fe Fe Mg Mg Ca Ca Na Na Cu Cu P P Mn Zn K - Cd - Pb - Vitamins C C E E A B1 - B2 - B3 Source: Mamadu et al., 2014

Contents Leaves Seeds Moisture 46.01 + 0.22 6.7 + 3 Protein 4.01 + 0.05 9.6 + 5 Fibers 12.36 +0.03 14.4 + 3 Ash 17.93 +0.04 12.7 + .6 Fat 12.02 + 0.05 5 + 0.05 CHO 7.67 + 0.03 58.1 Source: Warda et al., 2005

2.2. Phytochemical analysis Natural drugs have been a part of the evolution of human, healthcare for thousands of years. Nowadays nearly 88% of the global populations turn to plant derived medicines as their first line of defense for maintaining health and compacting diseases. One hundred and nineteen secondary plant metabolites derived from plants are used globally as drugs, 15% of all angiosperms have been investigated chemically and of that 74% of pharmacologically active plant derived components were discovered (Raja et al., 2009). 2.2.1 Procedure The plant samples were analyzed by the following procedures of Vinoth et al., (2012) to test for the presence of the main classes of phytochemicals individually. Saponins: Saponins were detected using the honeycomb froth test. Tannins: To a portion of the extract diluted with water, 3-4 drops of 10% ferric chloride solution is added. A blue color is observed for gallic tannins and green color indicates for catecholic tannins. Glycosides: About 25 ml of dilute sulphuric acid was added to 5 ml extract in a test tube and boiled for 15 minutes, cooled and neutralized with 10% NaOH, then 5 ml of Fehling solution added. Glycosides are indicated by a brick red precipitate. Alkaloids: About 2 ml of extract was measured in a test tube to which picric acid solution was added. An orange coloration indicated the presence of alkaloids. Flavonoids: About 4 ml of extract solution was treated with 1.5 ml of 50% methanol solution. The solution was warmed and metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was added and red color was observed for flavonoids and orange color for flavones. Volatile oils: About 2 ml of extract was shaken with 0.1 ml dilute NaOH and a small quantity of dilute HCl. A white precipitate is formed if volatile oils are present. Terpenoids: Four milligrams of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and red violet color was observed for terpenoid. The results of the qualitative phytochemical analysis by using different solvents were presented in Table (2.2).

Table (2.2): Qualitative phytochemical analysis by using different solvents

Solvents used for Acetone Methanol Ethanol extraction Alkaloid - - - Reducing sugar + + + Flavonoids - - + Saponnins + - + Tannins - - + Volatile oils - - - Glycoside + + - Terpenoids - + - Source: Samineh et al., 2013

2.3 Thin layer chromatography In thin layer chromatography, solid phase (i.e. absorbent) is coated with a Silica or Alumina. The coating acts as a binder on one side. The solid phase used may be glass, aluminum or plastic. A thin film of silica gel is coated on the glass plate. The mixture where to separate to spots is dissolved completely using any polar, non-polar, or acids. A mark is made by a pencil about 1cm height from the lower edge of the end of plate. The dissolved liquid of dry latex is spotted on this line, by the help of fine capillary, 2 or more spots can be placed on this line, but the distance between the two spots should be at least 2 cm. This distance maintains the spots from over lapping or mixing while separating. A solvent or mixture is selected and kept in a wide mouth container having a top cover to it. The plate is placed in this container such that the liquid solvent does not touch the line marked with spot with a pencil. This solvent (eluant) is flows and goes up by capillary action on the plate and it usually takes the compound present in the spot on the line to some distance and the compounds separate and appear as spots. Sometimes these spots appear as greasy appearance, yellow, white, brown or even red. Different spots are seen at different distance from the line marked. The number of spots that come on the silica plate depends on the solvent used, and number of compounds present in the dissolved liquid to be spotted on the line on the plate. Visualization of colored compounds is simple – the spots can be directly observed after development. Because most compounds are colorless however, a visualization method is needed. The silica gel on the TLC plate is impregnated with a fluorescent material that glows under ultraviolet (UV) light. A spot will interfere with the fluorescence and appear as a dark spot on a glowing background. While under the UV light, the spots can be outlined with a pencil to mark their locations. A second method of visualization is accomplished by placing the plate into iodine vapors for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is good practice to use at least two visualization techniques in case a compound does not show up with one particular method. The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the distance traveled by the substance divided by the distance traveled by the solvent. It's value is always between zero and one (Vishwa, 2014).

Chapter Three Materials and Methods

3.1 Materials The samples of Cassia senna (Sanamaka) leaves and seeds were brought from Wad Medani Town, Gezira State and from Erkweit area, Red Sea State in September 2014. The samples of both areas were shade dried, and then were transferred to Food Analysis Laboratory, Faculty of Engineering and Technology, University of Gezira, where the study tests were run. 3.2. Proximate analysis tests 3.2.1. Ash content The various samples were analyzed for their ash content by the procedure described by AOCS (1985), five grams of ground sample were weighed into previously heated, pre-dried and pre-weighed crucible. This crucible with its contents was then placed in a muffle furnace at 550℃ and maintained at this temperature for 5 hours. The crucibles were then transferred to a desicator, cooled at room temperature and reweighed. Ash content was then calculated on dry matter basis as follows: (푏 − 푎) × 100 100 퐴푠ℎ% = × 푀 (100 − 퐹) Where: a = weight of empty dish b = weight of dish with ash M = weight of sample (gm) F = moisture content of the sample 3.2.2. Oil content Oil contents of the various samples were determined according to AOCS (1985). In this method, three grams of ground sample were weighed into filter paper folded in such a way so as to prevent the escape of the meal. A piece of absorbent cotton was placed on the top of the thimble to distribute the solvent as it drops on the sample. The wrapped sample was then placed in the extraction tube of the soxhlet and then 150 ml of n-hexane (99% purity) was poured in the extraction flasks before fixing the tubes, after 6 hours of extraction the extraction flasks were disconnected. The hexane was recovered by distillation under vacuum. Last traces of hexane were

19 removed by putting the flasks in the oven. The flasks were then cooled at room temperature in a desicator and weighed. Oil content was calculated as follows: 푤푒𝑖푔ℎ푡 표푓 표𝑖푙 × 100 표𝑖푙% = 푤푒𝑖푔ℎ푡 표푓 푠푎푚푝푙푒 푤푒𝑖푔ℎ푡 표푓 표𝑖푙 × 100 표𝑖푙% (표푛 퐷. 푀 푏푎푠𝑖푠) = 푤푒𝑖푔ℎ푡 표푓 푠푎푚푝푙푒 × 퐷. 푀. % 3.2.3. Protein content Protein content of each tested sample was determined according to AACC (1983) in which one gram sample was digested using 25 ml of concentrated sulfuric acid for 6 hours. Then the digested sample was transferred to 100 ml volumetric flask. Five ml of the clean digested sample was pipetted into a distillation unit and then 10 ml of 40% NaOH was poured in the funnel. The ammonia trapped in boric acid (2%) was titrated against 0.1N HCl solution, a faint pink color was taken a the end point. The protein percentages were calculated as follows: 푚푙 표푓 0.1푁 퐻퐶푙 × 0.014 × 6.25 × 100 푐푟푢푑푒 푝푟표푡𝑖푒푛% = 푤푒𝑖푔ℎ푡 표푓 푠푎푚푝푙푒 3.2.4. Crude fiber content Crude fiber contents were determined for the various samples according to AOCS (1985). Three grams of the defatted sample were weighed into a 600 ml beaker. Then 200 ml of boiling 1.25% sulfuric acid and 1 drop of diluted antifoam agent were added. The contents were boiled under reflux for 30 minutes and filtrated through Buchner funnel. The residue were then transferred back into the beaker using 200 ml of 1.25% boiling sodium hydroxide, and boiled under reflux for 30 minutes. The contents were again filtered and transferred to a pre-dried and weighed dish. It was then dried at 100℃ to constant weight. The contents were then reweighed and ignited in muffle furnace at 550℃ for 5 hours. The crude fiber content was calculated as follows: (푎 − 푏) × 100 100 퐶푟푢푑푒 푓𝑖푏푒푟% (표푛 퐷. 푀. 푏푎푠𝑖푠 = × 푤 (100 − 푀) Where: a = weight of dish and content before ashing b = weight of dish and content after ashing M = moisture content W = weight of sample

3.2.5. Carbohydrates content Carbohydrates of both samples leaves and seeds (of both areas) were obtained by subtraction. % Carbohydrates content = 100- (%protein + % oil + % fiber + % ash). 3.3. Phytochemical screening tests Phytochemical screening for the presence of the main classes in the leaves and seeds of C. senna from two areas were done according to Yusha’u et al., (2009): 3.3.1 Test for the presence of alkaloids To about 0.1 ml of the extract and fractions in a test tube, 2 – 3 drops of Dragendoff’s reagent was added. An orange red precipitate with turbidity denoted the presence of alkaloids. 3.3.2 Test for the presence of flavonoids and flavonones To about 4 mg/ml of the extracts and fractions a piece of magnesium ribbon was added followed by drop-wise addition of concentrated HCl. A colour change from orange to red indicated the presence of flavonones; red to crimson indicated the presence of flavonoids. 3.3.3 Test for the presence of glycosides

Ten ml of 50% H2SO4 was added to 1 ml of the filtrate in separate test tubes and the mixtures heated for 15mins followed by addition of 10 ml of Fehling’s solution and boiled. A brick red precipitate indicated presence of glycosides. 3.3.4 Test for the presence of saponins To about 0.5 g of the powdered leaf was dispensed in a test-tube and 5.0 ml of distilled water was added and shaken vigorously. A persistent froth that lasted for about 15 minutes indicated the presence of saponins. 3.3.5 Test for the presence of steroids Two ml of the extracts were evaporated to dryness in separate test tubes and the residues dissolved in acetic anhydride followed by addition of chloroform.

Concentrated H2SO4 was added by means of a pipette via the side of the test tubes. Formation of brown ring at the interface of the two liquids and violet colour in the supernatant layer denoted the presence of steroids.

3.3.6 Test for the presence of tannins Two ml of the extract/fraction was diluted with distilled water in separate test

tubes; 2 – 3 drop of 5% ferric chloride (FeCl3) solution was added. A green – black or blue colouration indicated tannins. 3.4. Thin layer chromatography test In this test, the procedure described by Vogel et al., (2003) was carefully followed to run a test for the polar extract (methanolic extract) and the apolar extract (hexane extract) for the leaves and seeds of C. senna from two areas. Identification of the individual components of the crude polar or apolar by thin layer chromatography started by dissolving of about 5 ml of the crude extract in a small volume in methanol or n-hexane, respectively. The solution was applied as a band, using a micro-syringe on a TLC plate coated with silica gel (0.5 mm thickness). The plate was developed in a tank containing the solvent mixture (chloroform: methanol; 2:1) for about 45 minutes. After solvent drying at room temperature, the developed plate was covered with another clean glass plate exposing a silica gel zone of about 2 cm at one edge, the two plates were clamped together and a provision was made to ensure that only this narrow zone was been reached by the detection reagent. 3.5. Toxicity test of C. senna leaves and seeds powder Twenty of mosquitos' larvae (either Anopheles or Culex) were put in a beaker containing 250 ml water. 0.6 g of dried powder of leaves and seeds from each area were thrown gently and individually on those beakers. Each test was replicated twice. After 24 hours the dead larvae were counted as percentage mortalities. A control batch was usually designed and was used to correct the tested mortalities. The toxicity of leaves and seeds of C. senna from both areas were evaluated against both larvae of Anopheles and Culex mosquitoes. 2.6. Statistical analysis Microsoft Office, Excel 2007, was used to analyze the data obtained. Simple descriptive statistics (mean and standard deviation SD), simple regression analysis and ANOVA test, were used to describe the observed variation between the control and the irradiated samples of both plants used. The difference will be significant whenever the -stat value is bigger than that of -crit.

Chapter Four Results and Discussion

4.1 Proximate analysis of C. senna of Wad Medani and Erkweit areas The comparative nutritional composition, qualitative phytochemical screening, thin layer chromatography and toxicity, for Wad Medani (W) and Erkweit (E) samples of Cassia senna seeds (S) and leaves (L) were the aims of this study. The values of protein, crude fiber, ash, fat and carbohydrates in C. senna were represented in Table (4.1). The highest protein content was found in Erkweit seeds (19.34%), whereas the lowest protein value (17.21%) was noticed in Erkweit leaves. This implies that the amount of protein constituent elements is different in the two regions, as well as to the quality of fertilizer which added to the soil. The values of the two samples were higher than the value (4.01%) reported by Mubarak (2005). The highest crude fiber content (0.12%) was recorded in Wad Medani leaves, whereas the lowest fiber value (0.06%) and ash value (4.5%) were recorded in Wad Medani Seeds. The highest ash content (11.2%) was noticed in Erkweit leaves, which is an indication of high minerals content in Erkweit soil. The two samples quantities were lower comparable with the value of 17.93% reported by Mubarak, (2005), while the highest fat (0.91%) and carbohydrates contents were noticed in Wad Medani leaves and seeds, respectively. Lipids important in diets because it promotes soluble vitamins and does not add the bulk of the diet (Bogret et al., 1994). The carbohydrate plays a significant role in protein sparing action. Protein performs specialized function in the body building and growth. Carbohydrate comes to rescue and spare of proteins from being misused for caloric value (Mubarak, 2005). The statistical analysis revealed that, no significant differences between the four samples in their nutritional contents (f= 0.22; f-crit= 3.26). Although Anova proved a non-significant differences between the samples, but a fraction variation in any component could make a difference in any potential characteristics. Warda et al., 2005, who investigated the chemical and mineral contents in Cassia obovata from Western Sudan, found that, the protein contents were 4.01 and 9.60, the fiber contents were 12.36 and 14.4, the ash contents were 17.93 and 12.7, the fat contents were 12.02 and 5.0, and the carbohydrates contents were 7.67 and 58.1, respectively for leaves and seeds.

Table (4.1) The proximate analysis of C. senna brought from Wad Medani and from Erkweit areas

Contents Erkwit samples Wad Medani samples Leaves Seeds Leaves Seeds Protein 17.21 19.34 17.17 18.00 Fiber 0.08 0.09 0.12 0.06 Ash 11.20 5.25 7.80 4.50 Fat 0.16 0.10 0.91 0.08 CHO 54.75 61.42 64.20 66.62

Variance Average Sum Count SUMMARY 1.03 17.93 71.72 4 Protein 0.001 0.09 0.35 4 Fiber 9.15 7.19 28.75 4 Ash 0.16 0.31 1.25 4 Fat 26.28 61.75 246.99 4 CHO

ANOVA F crit P-value F MS df SS Source 3.26 5.71E-12 306.92 2662.97 4 10651.87 Rows 3.49 0.8805 0.22 1.91 3 5.73 Columns 8.68 12 104.12 Error

19 10761.72 Total

4.2. Qualitative phytochemical screening of C. senna The qualitative phytochemical screening of C. senna leaves and seeds that were brought from Wad Medani area and from Erkweit area, were presented in Table (4.2). Cassia senna leaves from Erkweit (EL) contained glycosides, flavonoids, flavonone and alkaloids, as same as ES (except that, ES contained also sterols). WL in their phytochemical contents resembles completely ES, but it differs from the sample WS from the same area in that, WS, lack of sterols and glycosides. According to Kumar et al., 2010, ES and WL possessed high antimicrobial, antithelmintic and antidiarrhoeal activity, whereas, less activity was expected from EL, while WS was at the last rank. Mamadu et al., 2014, run a phytochemical screening for leaves and seeds of C. senna, and he found that, the leaves contained alkaloids, anthraquinones, triterpenoids, flavonoids, steroids and glycosides, whereas, the seeds contained alkaloids, anthraquinones, and steroids only. Smith (2009) also runs a phytochemical screening for Cassia siamea from Pakistan. The results of his study revealed that, saponins, anthraquinones, tannins, alkaloids and oxalate were present in the ethanolic extract of the whole plant.

Table (4.2) Qualitative phytochemical screening of C. senna leaves and seeds

Contents EL ES WL WS Flavonoids + + + + Flavonone + + + + Tannins - - - - Saponins - - - - Alkaloids + + + + Sterols - + + - Glycosides + + + -

+ : mean presence of the main class - : mean absence of the main class

4.3. Thin layer chromatography test for C. senna A thin layer chromatography tests were run for the C. senna leaves and seeds (Table, 4.3) that were brought from Wad Medani area, Gezira State and from Erkweit area, Red Sea State. The tests involved the polar (ethanol) extract and the apolar (petroleum ether) extract for both leaves and seeds. The samples of Wad Medani separated two spots from the apolar extract of the leaves and only one spot from the polar extract of the leaves. Concerning seeds, the polar extract separated only one spot (as same as the polar extract of the leaves part), while the apolar extract separated three spots. A total of three spots were separated from the leaves and four spots were separated from the seeds of Wad Medani samples. The case was not similar for Erkweit samples, leaves part separated four spots (two from the polar and two from the apolar extracts), while that, five spots were separated from the seed part (two spots from the apolar extract and the rest from polar extract). The Rf values of the detected spots were not identical between the samples of the two areas and also between the two plant parts (leaves and seeds). Plants are potent biochemists and have been components of phytomedicine since times immemorial; man is able to obtain wondrous assortment of industrial chemicals. Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds (i.e. any part of the plant may contain active components). The systematic screening of plant species with the purpose of discovering new bioactive compounds is a routine activity in many laboratories. Plants are collected either randomly or by following local healers in geographical areas where the plants are found (Parekh et al., 2006). This study showed no similarity between the four samples in their bioactive compounds. These results indicate there's a difference in the biotic and a biotic environmental factors of the two regions and hence, doesn't reflect similarity of compounds from each part of the two plants. The concentrations of these compounds were consequently expected to be not similar, and this may be due to the weather, pH of the soil, minerals and some internal factors.

Table (4.3) Thin layer chromatography test (Rf values) for C. senna leaves and seeds brought from Wad Medani area and Erkweit area a) Wad Medani sample Spot No. Leaves Leaves Seeds petroleum Seeds ethanol petroleum ether ethanol ether 1 0.25 0.17 0.29 0.22 2 0.34 - 0.33 - 3 - - 0.46 - b) Erkweit sample Spot No. Leaves Leaves Seeds petroleum Seeds petroleum ether ethanol ether ethanol 1 0.36 0.34 0.61 0.30 2 0.59 0.53 0.84 0.50 3 - - - 0.69

4.4. Toxicity of C. senna leaves and seeds against mosquito larvae The toxicity of 0.6 g (per 250 ml water) powder of Cassia senna leaves and seeds which were brought from Wad Medani and Erkweit areas toward Anopheles and Culex larvae, was presented in Table (4.4). Concerning Erkweit samples, the leaves produced 100% mortality in Anopheles and Culex larvae, whereas, the seeds produced 45% mortality in Anopheles and 25% mortality in Culex. It seemed that, Anopheles larvae were more susceptible toward C. senna leaves than Culex larvae. Concerning Wad Medani samples, the leaves produced 80% mortality in Anopheles and 60% in Culex larvae, while that, the seeds produced 40% mortality in Anopheles and 37.5% mortality in Culex. It seemed that, Anopheles larvae were more susceptible toward C. senna leaves and seeds than Culex larvae. The analysis revealed that, C. senna could produce a mean of about 66.25% mortality in Anopheles larvae and 55.63% mortality in Culex larvae. Also, the leaf part, from both areas could produce high mortality (100% and 70%) than seed part (35% and 38.75%, respectively for Erkweit and Wad Medani samples). ANOVA, also proved that, the toxicity of the used plant part were statistically not similar (f=31.27; f-ctir=9.28, hence, the difference is significant). The reason for the difference in toxicity of C. senna parts could be attributed to their chemical and phytochemical constituents in addition to the susceptibility of both mosquito larvae. It was proved that, the leaves of Erkweit (EL) sample, contained glycosides, flavonoids, flavonone and alkaloids, as same as ES (except that, ES contained also sterols). Wad Medani leaves sample (WL), contents resemble completely ES, but WS from the same area, lack of sterols and glycosides. The TLC tests could also confirm the reason, in addition to predict the toxicity of the obtained spots. A total of three spots were separated from the leaves and four spots were separated from the seeds of Wad Medani samples, but the case was not similar for Erkweit samples, leaves part separated four spots (two from the polar and two from the apolar extracts), while that, five spots were separated from the seed part (two spots from the apolar extract and three from polar extract). The variations of toxicity of different extracts or the susceptibility of Anopheles and Culex larvae, were well reported by several researchers e.g. Yousif (2013). Govindarajan et al., (2005) and Sukumar et al., (1991) also introduced studies to determine the toxicity of Cassia senna against Anopheles and Culex larvae.

Table (4.4) Toxicity of 0.6 g (per 250 ml) powder of Cassia senna leaves and seeds (from Wad Medani and Erkweit areas) toward Anopheles and Culex larvae

Erkweit samples Wad Medani samples

Larvae Leaves Seeds Leaves Seeds

Anopheles 100 45 80 40 Culex 100 25 60 37.5 *- Mortality in control batch was zero

Variance Average Sum Count SUMMARY 822.92 66.25 265 4 Anopheles 1084.89 55.63 222.5 4 Culex

0 100 200 2 EL 200 35 70 2 ES 200 70 140 2 WL 3.125 38.75 77.5 2 WS

ANOVA Source of F crit P-value F MS df SS Variation 10.13 0.145 3.82 225.78 1 225.78 Rows 9.28 0.009 31.27 1848.69 3 5546.09 Columns 59.11 3 177.34 Error

7 5949.22 Total

Globally, there is a prompt awareness going on and always desired to use natural, ecofriendly compounds for larvicidal activity (Vinayagam and Senthil, 2008). Mosquito risk has become more acute in recent time and the death of millions of people every year due to mosquito-borne diseases has resulted in the loss of socioeconomic wealth in many countries (Pavela, 2008). Malaria remains a major health problem in Sudan. About 20 % 40% of outpatient clinic visits and approximately 30% of total hospital admissions are due to Malaria (WHO and UNICEF, 2005). Plant offer and alternative source of insect control agents because they contain a range of bioactive chemicals (Hedlin et al., 1997). Cassia senna is shrub or small tree widely spread in short grass Savannah throughout Central and Southren Sudan. Decoction of stem bark is used to treat Malaria (Musa et al., 2011). Hence, there is a need for developing biologically active natural chemical constituents which act as a larvicidal and promising to reduce the risk to humans and harmful accumulated residues (Prabakar and Jebanesan, 2004). This has necessitated need for research and development of environmentally safe, biodegradable, and low cost indigenous method for vector control, which can be used with minimum care by individuals and communities in specific situation (Kumar et al., 2012). The extract which is obtained from plant parts like leaves, root, flower, bark, seed, and fruits in their crude extracts has been used as conventional larvicide (Vinayagam and Senthil, 2008). The secondary compounds of plants are vast repository of compounds with a wide range of biological activities (Tennyson et al., 2012). Mosquito is the most indisputable significant arthropod vector of diseases. The borne diseases caused by mosquito are one of the major health problems in most of countries. It's affecting the socioeconomical status of many nations and it's an important pest against human causing diseases (Govindarajan et al., 2005). The control of mosquito by chemical substance is not safe at present because of insecticide resistance by vector and environmental imbalance. Application of chemical or synthetic insecticide lead to deleterious effects in long time, hence it doesn't provide absolute results. That is why alternative mosquito control method is needed (Pavela, 2008).

Chapter Five Conclusions and Recommendations

5.1 Conclusions: 1- The highest nutritional contents in all samples was the carbohydrates (61.75%) followed by protein (17.93%), then ash (7.19%), fat (0.31%) and lastly fibers (0.09%). Anova revealed that, no significant differences between the four samples in their nutritional contents. 2- The phytochemical screening, proved that the leaves of Erkweit (EL) sample, contained glycosides, flavonoids, flavonone and alkaloids, as same as ES (except that, ES contained also sterols). Wad Medani leaves sample (WL) in their phytochemical contents resemble completely ES, but WS from the same area, lack of sterols and glycosides. 3- A total of three spots were separated from the leaves and four spots were separated from the seeds of Wad Medani samples, but the case was not similar for Erkweit samples, leaves part separated four spots (two from the polar and two from the apolar extracts), while that, five spots were separated from the seed part (two spots from the apolar extract and three from polar extract). 4- C. senna produced a mean mortality in Anopheles more than in Culex larvae. Also, the leaf part, from both areas produced high mortality in mosquito larvae than seed part. ANOVA, proved that, the toxicity of the plant parts were statistically not similar.

5.2 Recommendations: 1- To get benefit of the nutritional contents from C. senna, either from Wad Medani or from Erkweit, should not be ignored. 2- The pharmaceutical uses of such plant should be tested. 3- Further analysis should be run to widen the knowledge about the constituents of C. senna leaves and seeds, from both areas.

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