Phytochemical and Biological Study of Tribulus Terrestris and Fagonia Arabica

Phytochemical and Biological Study of Tribulus terrestris and Fagonia arabica

A Thesis Submitted by Dina Magdy Abd El-Hameed Ghanem (Bachelor Degree of Pharmaceutical Sciences) Research Assistant – Pharmacognosy Department National Research Centre

For the Degree of Master in Pharmaceutical Sciences (Pharmacognosy)

Under the Supervision of

Prof. Dr. Seham Salah El-Din Prof. Dr. Nagwa Mohammed El-Hawary Ammar Prof. of Pharmacognosy Prof. of Pharmacognosy Department of Pharmacognosy Department of Pharmacognosy Faculty of Pharmacy National Research Centre Cairo University, Egypt Giza, Egypt

Pharmacognosy Department Faculty of Pharmacy Cairo University Egypt 2016

دراسة فيتوكيميائية و بيولوجية للتريبيوالس تريسترس و الفاجونيا ارابيكا

رسالة مقدمة من دينا مجدى عبد الحميد غانم )بكالوريوس العلوم الصيدلية( مساعد باحث- قسم العقاقير المركز القومى للبحوث

للحصول على درجة الماجستير فى العلوم الصيدلية )عقاقير(

تحت اشراف

أ.د. /سهام صالح الدين الهوارى أ.د. نجوى محمد عيسى عمار أستاذ العقاقير- قسم العقاقير أستاذ العقاقير- قسم العقاقير كلية الصيدلة – جامعة القاهرة المركز القومى للبحوث – الجيزة ج. م . ع ج. م . ع

قسم العقاقير كلية الصيدلة – جامعة القاهرة ج. م . ع ٦١٠٢

English Abstract

This study focused on two plants growing in Egypt, the aerial parts of Tribulus terrestris L. and Fagonia arabica L. (Family Zygophyllaceae), which have been used extensively in folk medicine for a variety of purposes. This study aimed for the performance of phytochemical and biological studies of the two plants under investigation.

Tribulus terrestris L. possesses many important biological activities; aphrodisiac activity, antimicrobial, cytotoxic and antioxidant activities. Fagonia arabica L. has effects on hematological parameters, as well as, antimicrobial, cytotoxic and antioxidant activities. The results of the biological study revealed that the aqueous methanolic extracts of the aerial parts of T.terrestris L. and F.arabica L. were responsible for these biological activities. So it was of deemed interest to investigate the phytoconstituents of the two bioactive extracts separately.

Phytochemical and chromatographic studies were performed using different solvent systems and detection by Khagi-Mischner and AlCl3 reagents which revealed the presence of high percentage of saponins and phenolic compounds in the two extracts under investigation. Therefore, it was decided to perform quantitative estimation of total flavonoid and phenolic contents, qualitative and quantitative estimation of flavonoids as well as phenolic compounds by HPLC analysis, isolation and identification of flavonoidal compounds, preparation and phytochemical investigation of saponin content through chromatographic and mass screening studies and finally quantitative estimation of diosgenin in T.terrestris L. by HPLC analysis.

The flavonoid content of the methanolic extracts of the aerial parts of T.terrestris L. and F.arabica L. were isolated where the extracts were subjected to fractionation using several chromatographic techniques (CC and TLC) leading to the isolation of five compounds which are identified using several spectroscopic techniques (UV, 1HNMR and 13CNMR). From T.terrestris L. quercetin and rutin and from F.arabica L. quercetin, quercetrin and naringenin were isolated respectively.

All the obtained results confirm the advantage of the use of T.terrestris L. and F.arabica L. as effective therapeutic agents from botanical origin. This can be realized after doing the necessary toxicological and pharmaceutical studies. The pharmaceutical formulation of the biologically active fractions is recommended after the toxicological tests. The teamwork is looking to achieve the benefits from this work by applying the primary clinical application of the promising results after doing the necessary toxicological and pharmaceutical studies.

Introduction

The genus Tribulus of the Zygophyllaceae family comprises several species which grow as shrubs or herbs in subtropical areas around the world (Achenbach et al., 1994). From which Tribulus terrestris L. is very common in Egypt. T.terrestris L. is very common in the Nile delta including Cairo but not further South, Nile valley from Cairo to Wadi Halfa, the Mediterranean coastal strip from El-sallum to Rafah, Sinai proper i.e. South of El-Tih desert (especially in waste places) (Täckholm, 1974). This plant can be found in arid climate regions around the world; for example, in Southern USA, Mexico, Spain, Bulgaria, India and China (Ganzera et al., 2001). On the other hand T.terrestris L. used in folk medicine against various diseases. The fruits and seeds are of great importance in folk medicine because they are used as an aphrodisiac, diuretic and anthelmintic, as well as, to treat cough and kidney failure. T.terrestris L. has the reputation of having anabolic effect as well as in USA as dietary supplement. It is used in many countries against sexual impotency, oedema, abdominal distention and cardiovascular diseases (Combarieu et al., 2003). Preparation containing T.terrestris L. extract are on sale in USA as food supplement with claim of general stimulating action on motor activity, muscle tone and restorative tonic for vigor. Indeed, T.terrestris L. preparations are mainly used to improve performance in sports and for treatment of impotency. More recently, it has been reported that an aqueous extract of T.terrestris L. appeared to possess aphrodisiac properties probably due to androgen increasing properties. Earlier investigation performed on T.terrestris L. resulted in the isolation of steroidal saponins, flavonoids, carbohydrates and alkaloids (De Kock and Enslin, 1958), (Tomova et al., 1974), (Perepelitsa and Kintya, 1975), (Hamed et al., 2004), (Wang and Lu, 1991), (Kang et al., 2014), (Bhutani et al., 1969), (Saleh et al., 1982), (Shi et al., 1999), (Reshma et al., 2015), (Borkowski and Lutomski, 1960), (Wu et al., 1999), (Sharma and Narula, 1977), (Huang et al., 1991) and (Chen et al., 2002).

The genus Fagonia (family Zygophyllaceae) is represented in Egypt by eighteen species (Täckholm, 1974). The Fagonia arabica – complex is made up of four species: F.arabica L., F.acerosa Boiss., F.zilloides Humbert and F.taeckholmiana Hadidi. While Fagonia acerosa Boiss. is only reported

5 from South Persia and West Pakistan on the Eastern distribution limits of F.arabica L., on the other hand, F.zilloides Humbert is only known from South Morocco and Fagonia taeckholmiana Hadidi is endemic to the Galala Desert of Egypt. F.arabica L. is considered to be wide spread, ranging in distribution from the North Africa Sahara to South Arabica and Pakistan (Hadidi, 1972). F.arabica L. and F.taekholniana Hadidi are found in Egypt. F.arabica L. is a common and rather polymorphic species with distinct spiny stipules, lower tri and upper unifoliate leaves. Fagonia taekholmiana is a rare species characterized by the absence of the spiny stipules and the distinctly unifoliate leaves (Täckholm, 1974). In traditional medicine, aqueous extracts of Fagonia species were reported to exhibit medicinal properties and used for treatment of cholera, prolonged fever and snake bites (Chopra et al., 1956). It’s reputed to be a medicinal plant in scientific and folkloric literature and its medicinal values are well documented (Chopra et al., 1982). An aqueous decoction of the aerial parts is a popular remedy in the indigenous system of medicine for cancer in its early stages and for treatment of various other diseases of digestive and blood vascular system (Saeed, 1969). The medicinal properties of the plant were attributed due to its variety of active constituents. Although the plant had received a great interest for the phytochemical investigation since many years, yet the chemical structure of most of its constituents were established during the last 15 years. The entire plants of various Fagonia species were investigated mainly for the presence of two major classes of phytochemical compounds, i.e. flavonol glycosides and terpenoid glycosides (Saleh et al., 1990), (Miyase et al., 1996), (El-Wakil, 2007), (El-Negoumy et al., 1986) and (El-Hadidi et al., 1988).

This study will include: Part I: Phytochemical study: 1. Preliminary phytochemical screening and proximate analysis of the aerial parts of T.terrestris L. and F.arabica L. 2. Investigation of the volatile constituents of the aerial parts of T.terrestris L. and F.arabica L. 3. Investigation of the lipoidal matter of the aerial parts of T.terrestris L. and F.arabica L. 4. Investigation of the carbohydrate content of the aerial parts of T.terrestris L. and F.arabica L.

5. Investigation of the protein and amino acid contents of the aerial parts of T.terrestris L. and F.arabica L. Part II: Biological study: 1. Determination of acute toxicity of T.terrestris L. and F.arabica L. 2. Evaluation of the aphrodisiac activity of T.terrestris L. 3. Evaluation of the effect on hematological parameters of F.arabica L. 4. Evaluation of the in vitro antimicrobial activity of T.terrestris L. and F.arabica L. 5. Determination of the in vitro cytotoxic activity of T.terrestris L. and F.arabica L. 6. Determination of the in vitro antioxidant activity of T.terrestris L. and F.arabica L.

Part III: Phytochemical investigation of biologically active extracts: 1. Phytochemical investigation of flavonoid and phenolic contents in the bioactive aqueous methanolic extract of T.terrestris L. and F.arabica L. 2. Investigation of the flavonoidal compounds in the bioactive methanolic extract of T.terrestris L. and F.arabica L. 3. Phytochemical investigation of saponin content in the bioactive aqueous methanolic extract of T.terrestris L. and F.arabica L.

Aim of work:

Our aim of work is phytochemical and biological studies of the successive extracts of Tribulus terrestris L. and Fagonia arabica L. (Family Zygophyllaceae), growing in Egypt, as well as the isolation and identification of the biologically active natural fractions or compounds isolated from the two plants under investigation, which can be useful in drug industry as curative or complementary medicine.

Summary

This work studied two plants T.terrestris L. which possesses many important biological activities; aphrodisiac activity, antimicrobial, cytotoxic and antioxidant activities; and F.arabica L. which has effects on hematological parameters, as well as, antimicrobial, cytotoxic and antioxidant activities. The aim of this study is phytochemical and biological investigation of the two plants growing in Egypt.

The present work is divided into three parts: Part I: Phytochemical Study. This included: 1. Literature survey of the two plants under investigation including phytochemical constituents and biological activities. 2. Preliminary phytochemical screening and proximate analysis of the aerial parts of T.terrestris L. and F.arabica L. 3. Investigation of the volatile constituents of the aerial parts of T.terrestris L. and F.arabica L. 4. Investigation of the lipoidal matter of the aerial parts of T.terrestris L. and F.arabica L. 5. Investigation of the carbohydrate content of the aerial parts of T.terrestris L. and F.arabica L. 6. Investigation of the protein and amino acid contents of the aerial parts of T.terrestris L. and F.arabica L.

Part II: Biological Study. This included: 1. Determination of acute toxicity of successive extracts of T.terrestris L. and F.arabica L. 2. Evaluation of aphrodisiac activity of Tribulus terrestris L. 3. Evaluation of the effect on hematological parameters of Fagonia arabica L. 4. Evaluation of the in vitro antimicrobial activity of T.terrestris L. and F.arabica L. 5. Determination of the in vitro cytotoxic activity of T.terrestris L. and F.arabica L.

6. Determination of the in vitro antioxidant activity of T.terrestris L. and F.arabica L.

Part III: Phytochemical investigation of biologically active extracts. This included: 1. Phytochemical investigation of flavonoid and phenolic contents in the bioactive aqueous methanolic extract of T.terrestris L. and F.arabica L. 2. Investigation of the flavonoidal compounds in the bioactive methanolic extracts of T.terrestris L. and F.arabica L. 3. Phytochemical investigation of saponin content in the bioactive aqueous methanolic extracts of T.terrestris L. and F.arabica L.

Part I: Phytochemical study: 1. Preliminary phytochemical screening and proximate analysis of the aerial parts of T.terrestris L. and F.arabica L.: Preliminary phytochemical screening of the two plants under investigation was performed and revealed that the dried powdered aerial parts of the two plants contain: volatiles, sterols and/or terpenes, carbohydrates and/or glycosides, flavonoids (free and combined), coumarins, alkaloids and/or nitrogenous compounds and saponins. The yield of successive extractives with different solvents is variable; extracts with polar solvents are rich in phenolic compounds and saponins. While the non-polar extracts are rich in terpenoid and/or steroidal compounds. 1- Carbohydrates and/or glycosides are present in the methanol and the aqueous methanol extracts of the two plants under investigation. 2- Sterols and /or terpenes are present in the petroleum ether, diethyl ether, and chloroformic extracts of the two plants under investigation. 3- Saponins are detected only in the methanolic and aqueous methanol extracts of the two plants under investigation. 4- Flavonoids are appreciably present in the methanolic and aqueous methanol extracts of the two plants under investigation. 5- Anthraquinones and tannins are absent from the two plants under investigation.

2. Investigation of the volatile constituents of the aerial parts of T.terrestris L. and F.arabica L.: The volatile constituents of the fresh aerial parts of T.terrestris L. and F.arabica L. were prepared by hydro-distillation and were analyzed by GC/MS. The GC/MS analysis of volatile constituents of T.terrestris L. revealed that thirty two compounds out of forty four compounds were identified representing 72.73% of the total volatile constituents of the aerial parts of Tribulus terrestris L. Presence of a number of identified hydrocarbons and oxygenated compounds. The major compounds were methyl linolenate (18.59 %), 1,2,4-trimethyl benzene (9.56%), butylated hydroxytoluene (9.28 %), methyl palmitate (7.62 %) and 3,5,5- trimethylcyclohexene (7.48%). The analysis of the volatile constituents shows 32 components; oxygenated components were found to be the main constituents (53.15%) while the percentage of hydrocarbons reached (46.87%). Oxygenated monoterpenes were mainly dill ether (0.79 %) and oxygenated sesquiterpenes were mainly butylated hydroxytoluene (9.28%) and dihydroagarofuran (0.51%). Oxygenated diterpenes were mainly phytol (0.65%). Diterpene hydrocarbons were found mainly neophytadiene (2.53%) and eicosane (0.69%). Fatty acids and esters exhibited (37.07%) of the volatile constituents. The GC/MS analysis of volatile constituents of F.arabica L. revealed that twenty eight compounds were identified representing 96.6% of the total volatile constituents of F.arabica L. Presence of a number of identified hydrocarbons and oxygenated compounds. The major constituent of the oil was linalool (24.97%). The analysis of the essential oil shows 28 components; oxygenated components were found to be the main constituents (71.43%) while the percentage of hydrocarbons reached (28.57%). The major oxygenated monoterpenes were linalool (24.97%) and geraniol (6.48%) while the main oxygenatd sesquiterpene was β-eudesmol (3.45%). Sesquiterpene hydrocarbons were found mainly δ- cadinene (3.05%), and β-gauiene (1.1%). Fatty acids and esters exhibited (4.64%) of the volatile constituents. The major compounds are linalool (24.97%), phytol (8.5%), α-terpineol (4.45%), β-eudesmol (3.45%), α- damascone (3.24%) and δ-cadinene (3.05%).

3. Investigation of the lipoidal matter of the aerial parts of T.terrestris L. and F.arabica L.: The unsaponifiable and saponifiable matter of the aerial parts of T.terrestris L. and F.arabica L. were prepared and analyzed using GLC. The yield of lipoidal matter in T.terrestris L. is (2.78%) and in F.arabica L. is (1.3%) . The percentage of the USM fraction in T.terrestris L. is (48%) and in F.arabica L. is (78%). The percentage of total fatty acids fraction in T.terrestris L. is (46%) and in F.arabica L. is (28%). The percentage of hydrocarbons identified in the USM of T.terrestris L. is (38.596 %) and in

F.arabica L. is (59.375%). n-nonadecane C19 (8.8%) is the main hydrocarbon identified in the USM of T. terrestris L. and n-tricosane C23 (9.08%) is the main hydrocarbon identified in the USM of F.arabica L. The sterols identified in the USM of T.terrestris L. comprise: cholesterol (3.56%), campesterol (1.71%) and stigmasterol (1.08%). The triterpenoidal compound α-amyrine (5.65%) was identified in the USM of T. terrestris L. The sterols identified in the USM of F.arabica L. comprises: cholesterol (11.53%), campesterol (3.304%) and stigmasterol (0.803%). The triterpenoidal compound α-amyrine (18.627%) was identified in the USM of F.arabica L. The percentage of identified unsaturated fatty acids of T.terrestris L. is (28.68 %). linolenic acid is the major unsaturated fatty acid (17.6 %). The percentage of identified unsaturated fatty acids of F.arabica L. is (39.34 %). linolenic acid is the major unsaturated fatty acid (24.437 %). The percentage of identified saturated fatty acids of T.terrestris L. is (53.7%). heptadecanoic acid is the major saturated fatty acid (33.56 %). The percentage of identified saturated fatty acids of F.arabica L. is (20.17%). heptadecanoic acid is the major saturated fatty acid (14.82 %).

4. Investigation of the carbohydrate content of the aerial parts of T.terrestris L. and F.arabica L.: This includes the quantitative analysis of the carbohydrate content of the two plants under investigation, including both free sugars and polysaccharides. The free sugars of the aerial parts of T.terrestris L. and F.arabica L. were prepared and analyzed by HPLC for their identification. The HPLC analysis showed that major sugar in T.terrestris L. was raffinose (0.7 %) and in F.arabica L. was stachyose (0.47%) respectively. The polysaccharides of the aerial parts of T.terrestris L. and F.arabica L. were

12 prepared. Prepared polysaccharides were acid hydrolyzed and analyzed by HPLC for their identification and quantitative determination. The main polysaccharide hydrolysates of T.terrestris L. were inulin and arabinose (5.61% and 2.38% respectively) while in F.arabica L. the main polysaccharide hydrolysates were inulin and glucose (5.52 % and 1.02 % respectively).

5. Investigation of the protein and amino acid contents of the aerial parts of T.terrestris L. and F.arabica L.: The total protein content of the aerial parts of T.terrestris L. and F.arabica L. was determined using Kjeldahl method. Results revealed that the total protein content of the aerial parts of T.terrestris L. and F.arabica L. was 16.63% and 14.0%, respectively. The analysis of the amino acid content of the aerial parts of both plants under investigation revealed the presence of phenylalanine, threonine, valine, leucine and lysine as essential amino acids in the two plants with different proportions. The major essential amino acids in T.terrestris L. were leucine (0.5%) while in F.arabica L. was valine (1.2 %), respectively. The major non-essential amino acid in T.terrestris L. and F.arabica L. was glutamic acid (2.85% and 3.81%, respectively). Isoleucine (0.32%) and histidine (22%), essential amino acids were only present in F.arabica L. Cystin (2.42 %) and proline (0.20 %), non-essential amino acids, were only present in F.arabica L.

Part II: Biological study:

The successive extracts of the aerial parts of T.terrestris L. and F.arabica L. were used in the biological studies.

1. Determination of acute toxicity of successive extracts of T.terrestris L. and F.arabica L.:

Successive extracts of the aerial parts of T.terrestris L. and F.arabica L., were prepared and the acute lethal toxicity was done. The results showed that neither of the tested extracts of T.terrestris L. up to a dose of 1 g/kg mice body weight of the successive extracts and 10 mg/kg mice body weight of the prepared saponins caused any toxicity. Concerning F.arabica L. the results revealed that successive extracts up to a dose of 1

13 g/kg mice body weight of polar and nonpolar extracts and 20 mg/kg mice body weight of prepared saponins caused any toxicity.

2. Evaluation of aphrodisiac activity of T.terrestris L.: The aphrodisiac activity was carried out on the prepared successive extracts (petroleum ether, aqueous methanol and saponins extracts) of T.terrestris L. at doses of 5 mg/kg bodyweight. The results showed that in the present study oral administration of T.terrestris L. aqueous methanol extract and saponin fraction elevate testosterone and reduce LH plasma levels, which accompanied with significant elevation of prostate weight to reach normal levels.

3. Evaluation of the effect on hematological parameters of F.arabica L.: This study was carried out on the prepared successive extracts (polar, nonpolar and saponin extracts) of F.arabica L. in doses equal to 250 mg/kg rat body weight of polar and nonpolar extracts and 10 and 20 mg/kg rat body weight of saponin fraction. Hemoglobin and hematocrit % as indicator to hematological status showed non-significant changes in rats given oral administration of polar and non-polar extract of Fagonia arabica L. for three weeks, while reduced significantly in rats given oral administration of both saponin doses (10 and 20 mg/kg rat body weight). Plasma activities of ALT and AST as indicator to liver function showed non-significant changes in all rats groups. Kidney functions (plasma creatinine and urea) showed non- significant changes in all rats groups. The non-significant changes of liver and kidney functions in the present study revealed the complete safety of the studied extracts.

4. Evaluation of the in vitro antimicrobial activity of T.terrestris L. and F.arabica L.: The successive extracts of both plants were tested for their in vitro antibacterial and antifungal activities using the standard agar disc diffusion method against five bacterial species (Gram-positive: Bacillus sabtilis and Staphylococcus aureus, and Gram-negative: Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) and one fungal species (Candida albicans). Results showed that: T.terrestris L.

has more potent antimicrobial activity than F.arabica L. The chloroform extract of T.terrestris L. is the most powerful extract; it has the highest percentage of bacterial removal against the five bacterial strains tested. The F.arabica L. has limited antimicrobial activity. The methanol fraction of F.arabica L. is the most powerful fraction by comparing it to the other fractions. Against E.coli the most powerful fractions are chloroform extract of T.terrestris L., methanol extract of T.terrestris L., 50% aqueous methanol of T.terrestris L. and diethyl ether extract of T.terrestris L. which have percentage of bacterial removal equal to 93.87, 91.25, 91.00 and 90.00 respectively. Against Staph. aureus the most powerful fractions are chloroform extract of T.terrestris L., 50% aqueous methanol extract of T.terrestris L., diethyl ether extract of T.terrestris L. and diethyl ether extract of F.arabica L. which have percentage of bacterial removal of 88.51, 84.38, 68.5 and 63.01 respectively. Against Candida albicans the most powerful extracts are chloroform extract of T.terrestris L., methanol extract of F.arabica L., chloroform extract of F.arabica L. and methanol extract of T.terrestris L. which have percentage of bacterial removal of 93.39, 83.93, 62.50 and 60.26 respectively. Against Bacillus sabtilis the most powerful extracts are diethyl ether extract of T.terrestris L., chloroform extract of T.terrestris L., methanol extract of T.terrestris L. and methanol extract of F.arabica L. which have percentage of bacterial removal of 95.87, 80.22, 77.78 and 76.92 respectively. Against Salmonella typhimarium the most powerful extracts are 50% aqueous methanol extract of F.arabica L., chloroform extract of T.terrestris L., methanol extract of T.terrestris L. and diethyl ether extract of T.terrestris L. which have percentage of bacterial removal of 72.69, 72.30, 67.66 and 67.31 respectively.

5. Determination of the in vitro cytotoxic activity of T.terrestris L. and F.arabica L.: The successive extracts of both plants were tested for their in vitro cytotoxic activity using MTT assay. The most effective fractions against hepatocellular carcinoma (HepG2) are methanol extract of T.terrestris L., diethyl ether extract of F.arabica L., petroleum ether extract of T.terrestris L. and 50% aqueous methanol extract of F.arabica L. which

have LC50 equals to 16.1 µg/ml, 17.3 µg/ml, 21.5 µg/ml and 23.6 µg/ml respectively.

IC50 dose of different extracts of Fagonia arabica L. (polar, nonpolar and saponin) against MCF7 (breast carcinoma cell) was determined. The results revealed that polar extract and saponin showed anti-cancer activity against breast carcinoma cell MCF7. Saponin was

the most efficient where IC50 was 36.2g and IC90 was 61.8 g,

followed by polar extract (IC50 was 51.2 g and IC90 was 84.1 g). Non- polar extract was non-effective against breast carcinoma cell MCF7.

6. Determination of the in vitro antioxidant activity of T.terrestris L. and F.arabica L.: T.terrestris L. has higher antioxidant activity than F.arabica L. Polar extract and saponins extract of T.terrestris L. have the highest antioxidant activity which is 26.5% 18.5% respectively. Nonpolar extract and saponins extract of F.arabica L. has no antioxidant activity.

Part III: Phytochemical investigation of biologically active extracts:

1. Phytochemical investigation of flavonoid and phenolic contents in the bioactive aqueous methanolic extracts of T.terrestris L. and F.arabica L. Total phenolic content was determined using Folin-Ciocalteu colorimetric method. Results revealed that the total phenolic content of the aerial parts of T.terrestris L. and F.arabica L. was 11.160 and 9.415 mg (GAE)/g, respectively. Total flavonoidal content was determined according to Rolim et al., (2005). Results revealed that the flavonoidal content of the aerial parts of T.terrestris L. and F.arabica L. was 6.076% and 3.297%, respectively. Quantitative estimation of flavonoid and phenolic contents of the two plants under investigation by using HPLC revealed the presence of different compounds with different proportions. Quantitative estimation of rutin in the aqueous methanolic extract of T.terrestris L. and F.arabica L. using HPLC revealed that F.arabica L. contains higher percentage of rutin than T.terrestris L.

The percentage of rutin in T.terrestris L. and F.arabica L. was 0.006% and 0.013% respectively.

2. Investigation of the flavonoidal compounds in the bioactive methanolic extracts of T.terrestris L. and F.arabica L.: The flavonoid content of the methanolic extracts of the aerial parts of T.terrestris L. and F.arabica L. were isolated where the extracts were subjected to fractionation using several chromatographic techniques (CC and TLC) leading to the isolation of five compounds which are identified using several spectroscopic techniques (UV, 1HNMR and 13CNMR). From T.terrestris L. quercetin and rutin and from F.arabica L. quercetin, quercetrin and naringenin were isolated respectively.

3. Phytochemical investigation of saponin content in the bioactive aqueous methanolic extracts of T.terrestris L. and F.arabica L.:

- Isolation and quantitative determination of total saponin content of the aerial parts of T.terrestris L. and F.arabica L. was performed and the percentage was 7.38% and 9.66% respectively. - TLC and mass screening of the saponin hydrolysate revealed the presence of diosgenin in T.terrestris L. and presence of hederagenin and oleanolic acid in F.arabica L.. - Quantitative estimation of diosgenin in T.terrestris L. using standard diosgenin as a reference material was carried out and the result was 0.51%.

Conclusions:  T.terrestris L. and F.arabica L. proved remarkable and significant biological activities. Both plants under investigation have antimicrobial, cytotoxic and antioxidant activities. T.terrestris L. possesses a potent aphrodisiac activity while F.arabica L. has effects on hematological parameters and has cytotoxic effect against MCF7. Therefore an extensive toxicological study is needed to confirm the safety of the bioactive fractions or compounds.

 The pharmaceutical formulation of the biologically active fractions is recommended after the toxicological tests.

 The teamwork is looking to achieve the benefits from this work by applying the primary clinical application of the promising results after doing the necessary toxicological and pharmaceutical studies.