Cairo University Faculty of Pharmacy Pharmacognosy Department

Phytochemical and Bioactivity Studies on cairica (L.) Sweet and Jacquemontia pentantha (Jacq.) G. Don (Family ) A thesis submitted by Dina Mahfouz Eskander Research Assistant, Chemistry of Natural Compounds Department, National Research Centre, Cairo

for the PhD. Degree in Pharmaceutical Sciences (Pharmacognosy)

Under the Supervision of

Prof. Dr. Elsayed Ali Aboutabl Professor of Pharmacognosy Pharmacognosy Department Faculty of Pharmacy Cairo University Prof. Dr. Mahmoud Ibrahim Ali Nassar Professor of Natural Products Chemistry Chemistry of Natural Compounds Department National Research Centre, Cairo

Pharmacognosy Department Faculty of Pharmacy Cairo University 2016 Abstract

(Key Words: Jacquemontia pentantha - Ipomoea cairica - Convolvulaceae - DNA fingerprinting - proximate analysis – flavonoids – quantitative phenolic acids - HPLC profile - biological activities ).

This work includes phytochemical and bioactivity studies on Jacquemontia pentantha (Jacq.) G Don and Ipomoea cairica (L.) Sweet, family Convolvulaceae. Studying the following: literature survey of phytoconstituents and bioactivities of certain Convolvulaceae species, DNA fingerprinting, phytochemical screening and proximate analysis of both , HPLC profile of flavonoid compounds of both plants, quantitative estimation of phenolic acids and flavonoids of aerial parts of both plants and isolation, characterization and structure elucidation of the main constituents in the methanol soluble fraction of the aerial parts of Jacquemontia pentantha (Jacq.) G. Don .Also, evaluation of certain biological activities of extracts of the aerial parts of both plants including: determination of acute toxicity (LD50), anti-inflammatory activity, antihyperglycemic activity, antioxidant activity and hepatoprotective activity and also evaluation of antimicrobial activity of extracts of the aerial parts of both plants .

INTRODUCTION

Convolvulaceae, known commonly as the bindweed or morning glory family of flowering plants, includes 57 genera and about 1600 species, widely cultivated for their colourful funnel-shaped flowers and heart-shaped leaves. Most are twining and erect herbs, with a few woody , trees, and shrubs (Austin, 1973). In Egypt, Convolvulaceae is represented by 10 genera and 43 species (Boulos, 1983, Tackholm, 1974). The family is widespread in both tropical and subtropical areas (Meira et al., 2012). Convolvulaceae has many economic uses as, edible crops, ornamentals, medicinal plants, some serious weeds. It comprises important food plants e.g., the (Ipomoea batatas L.) and Forssk. It includes several ornamentals used in horticulture, several medicinal plants (Ipomoea, Cuscuta, Dichondra, Evolvulus) and numerous noxious weeds (Cuscuta and Convolvulus) (Rhui-cheng and Shu-huain, 1979). Secondary metabolites of Convolvulaceae may be accumulated in all parts of the or only in specific organs, e.g., seeds, leaves or roots (Eich, 2008). Phytoconstituents of many species of this family include; alkaloids (Leistner and Steiner, 2009), phenylpropanoids including flavonoids, as well as terpenoids and coumarins are constituents of plants of this family (Eich, 2008). In contrast, resin glycosides, characteristic constituents of complex resins occurring usually with lactifers, represent unique metabolites in the plant kingdom confined to the Convolvulaceae (Pereda- Miranda et al., 2010). These glycoresins constituents are responsible for the purgative properties of some species of the Convolvulaceae (Pereda-Miranda and Bah, 2003). Species of family Convolvulaceae are recieving increasing interest, as they are biologically active. Bioactivities, so far, detected in these species are antioxidant (Mahmood et al., 1993), antimicrobial (Panaccione, 2005), anti-inflammatory (Ferreira et al., 2006), antidiabetic (Jayaweera, 1982) and ( Malalavidhane et al., 2001) and anticancer activity (Cho et al., 2004). Accordingly; two plants Jacquemontia pentantha (Jacq.) G. Don and Ipomoea cairica (L.) Sweet belonging to family Convolvulaceae cultivated in Egypt were chosen for investigation of their phytoconstituents and bioactivities.

The aim of work :

A. Literature survey of phytoconstituents and bioactivities of certain Convolvulaceae species.

B. Identification of DNA Fingerprinting of Jacquemontia pentantha (Jacq.) G. Don and Ipomoea cairica (L.) Sweet leaves.

C. Phytochemical study of the aerial parts of each Jacquemontia pentantha (Jacq.) G. Don and Ipomoea cairica (L.) Sweet including the following:  Phytochemical screening and proximate analysis.  Quantitative estimation of phenolic acids and flavonoids.  HPLC profile of flavonoid compounds.  Isolation, characterization and structure elucidation of the main constituents in the methanol soluble fraction of the aerial parts of Jacquemontia pentantha (Jacq.) G. Don .

D. Evaluation of certain biological activities of extracts of the aerial parts of Jacquemontia pentantha (Jacq.) G. Don and Ipomoea cairica (L.) Sweet including:

 Determination of acute toxicity (LD50)  Anti-inflammatory activity  Antihyperglycemic activity  Antioxidant activity  Hepatoprotective activity  Antimicrobial activity

Review: 1. Phytoconstituents reported from certain Convolvulaceae species

Several constituents including alkaloids, flavonoids, phenolic acids, Phenolic acid derivatives, coumarins, triterpenes and sterols, lignans, fatty acids and resin glycosides are reported in different species of the family.

2. Bioactivities reported from certain Convolvulaceae species:

Allelopathic potential of compounds isolated from cav. Convolvulaceae, was studied by Anaya et al., 1990. was screened for inhibitory activity on AchE, the results obtained were very interesting for further isolation of acetylcholinesterase inhibitors, to be used in the treatment of Alzheimer’s disease (Feitosa et al., 2011). Bioactivity-guided purification of n-BuOH soluble fraction from the ethanol extract of Evolvulus alsinoides resulted in the isolation of two new compounds, 2,3,4-trihydroxy-3- methylbutyl 3-[3-hydroxy-4-(2,3,4-trihydroxy-2-methylbutoxy)- phenyl]-2-propenoate and 1,3-di-O-caffeoyl quinic acid methyl ester. Compound displayed most promising antistress effect by normalizing hyperglycemia, plasma corticosterone and adrenal hypertrophy. Compound was also effective in normalizing most of these stress parameters (Gupta et al., 2007). The antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative disease was studied. (Auddy et al., 2003). These plants including Evolvulus alsinoides are used for the management of neurodegenerative diseases such as Parkinson's, Alzheimer's, loss of memory, degeneration of nerves and other neuronal disorders by the Ayurvedic practitioners. Evolvulus alsinoides is well known for its memory enhancement, antiepileptic and immunomodulatory properties in the traditional Indian system of medicine. It exhibited adaptogenic and memory enhancing properties in rodents (Siripurapu et al., 2005). The antiseptic, anti-inflammatory, antibacterial, antispasmodic, antiviral and antifungal activities have been reported (Singhel et al., 2011) for Argyreia nervosa (Convolvulaceae). The use of Convolvulus arvensis as a candidate medicinal plant including anti-tumor, anti-angiogenesis and immunostimulatory activities (Mahmoudi et al., 2014). Antioxidant activity, antimicrobial effect, anthocyanin and phenolic content of purple sweet potato and its sections were evaluated. The high phenolic and anthocyanin content was responsible for its significant antioxidant activity (Cevallos- Casals and Cisneros-Zevallos, 2002).

Summary:

Convolvulaceae, known commonly as the bindweed or morning glory family of flowering plants, which includes 57 genera and about 1600 species, widely cultivated for their colourful funnel- shaped flowers and heart-shaped leaves. The family is widespread in both tropical and temperate areas. In Egypt , there are 10 genera and about 43 species of Convolvulaceae (Boulous , 1983) (Tackholm, 1974). Phytoconstituents of many species of family Convolvulaceae include; alkaloids, tannins,triterpenes, carbohydrates, flavonoids, sterols and coumarins. Two plants Jacquemontia pentantha (Jacq.) G. Don and Ipomoea cairica (L.) Sweet belonging to family Convolvulaceae were chosen to carry out phytochemical and bioactivity studies on them.

1. DNA Fingerprinting of Jacquemontia pentantha and Ipomoea cairica aerial parts

The genomic DNA of the two species has been subjected to RAPD and ISSR analyses. Eleven decamer olygonucleotide primers induced successive amplifications with a large number of polymorphic bands.The analysis of the amplicons generated by RAPD and ISSR reactions revealed that the biotypes of Jacquemontia pentantha and Ipomoea cairica species were reproducer and gave different molecular patterns. The banding patterns produced by the primers OP–AO2, OP – AO7, and HB–O9 generated the highest degree of polymorphism and induced unique fragments. Therefore, this primers could be used to discriminate between interspecific species. However, primer OP–AO1, HB-12 generated the lowest percent of polymorphism with consequent high percent of similarity coefficient.This primer could be used for the identification of the genotype. RAPD and ISSR technique are widely used to fingerprint and assess genetic diversity as previously reported by (Dalhaber et al., 2002, and Salem et al., 2007).

2. Preliminary Phytochemical Screening and Proximate Analysis

2.1. Preliminary Phytochemical Screening It revealed that the dried powdered aeriel parts of each of Jacquemontia pentantha and Ipomoea cairica contain carbohydrates and/ or glycosides, tannins, sterols and /or triterpenes, as well as flavonoids, while saponins being absent. Volatile oils, alkaloids and/or nitrogenous bases and coumarins are absent in Jacquemontia pentantha, while present in Ipomoea cairica.

2.2. Proximate Analysis

Proximate Analysis of air-dried aerial parts of Jacquemontia pentantha and Ipomoea cairica revealed moisture content of 8.77 and 8.63 %, total ash of 10.33 and 10.63 %, water-soluble ash of 5.28 and 3.81 %, acid-insoluble ash of 1.02 and 0.50 %, crude fiber content of 27.94 and 24.33 %, respectively.

3. Quantitative Estimation of Phenolic Contents of Jacquemontia pentantha and Ipomoea cairica Aerial Parts

3.1. Quantitative estimation of total phenolic and total flavonoid contents of Jacquemontia pentantha and Ipomoea cairica unflowering aerial parts

The total 70% methanolic extract, of both Jacquemontia pentantha and Ipomoea cairica unflowering aerial parts were chemically estimated. The content of total phenolics in the extracts was measured using FCR assay and is expressed as gallic acid equivalent (GAE), while the total flavonoids were estimated using aluminum chloride method (expressed as quercetin equivalent, QE). The total phenolic contents in Jacquemontia pentantha aerial parts was (8.49 ± 0.76) while flavonoid content was 6.22 ± 0.25. The total phenolic contents in Ipomoea cairica aerial parts was (9.57 ± 0.89) while, flavonoid content was 3.075 ± 0.33.

3.2. HPLC of flavonoid compounds of Jacquemontia pentantha and Ipomoea cairica aerial parts

The concentration of each of the identified component in methanol extracts was determined by HPLC analysis using external standard method. The identified flavonoid compounds in Jacquemontia pentantha and Ipomoea cairica extracts were ten compounds . Four of the identified flavonoid compounds from Jacquemontia pentantha: quercetin-3-glucoside, quercitrin, kaempferol and quercetin, were isolated as J - 5, J - 8, J - 9, and J -10 in this work with other flavonoid compounds. Quercetin-3- glucoside showed to be the most abundant flavonoid compound in Jacquemontia pentantha . While, two in Ipomoea cairica luteolin and apigenin . (Al-Okbi et al., 2010) . Also three other flavonoid compounds were previously isolated: quercetin 7-O-β- D – glucoside (Al-Okbi et al., 2010) ,

4. Isolation of Phytoconstituents: The study of the phytoconstituents of Jacquemontia pentantha aerial parts resulted in the isolation and identification of the following compounds:

1. Quercetin 3- O-(2-E-caffeoyl-ß-D-xylopyranosyl)–(1→2)–α– L–rhamno-pyranosyl–(1→2)– ß–D–glucopyranosid. 2. Isorhamnetin 3-O-β- D-glucoside. 3. Kaempferol 3-O-β-D-glucopyranoside (astragalin). 4. Kaempferol 3 –O-β-D- galactopyranoside. 5. Quercetin 3-O-ß-D-glucopyranoside. 6. Quercetin-3-O-β-galactopyranoside. 7. Quercetin 3-O-ß- xylopyranoside. 8. Quercetin 3-O-α-L- rhamnopyranoside. 9. Kaempferol. 10. Quercetin.

5. Bioactivity Evaluation: Bioactivity evaluation of Jacquemontia pentantha and Ipomoea cairica aerial parts were carried out on 70% methanol total extract and methanol fraction as follows:

5.1. Determination of Median Lethal Dose (LD50). Total extract 70% methanol of the aerial parts of Jacquemontia pentantha is safe up to 7.100 g /kg and up to 6.375 g /kg for Ipomoea cairica.

5.2. Antihyperglycemic Activity

The antihyperglycemic activity of the tested extracts aerial parts of Jacquemontia pentantha were tested after 2 and 4 weeks intervals. The 70% methanol total extract (100 mg/kg) showed a percentage of change in serum glucose level of 31.5 % and 58.04 % respectively. The reference drug metformin (100 mg/kg)showed a decrease in serum glucose level by 35.8 % and 67.3 % after 2 and 4 weeks respectively.The percentage of change of 70% methanol total extract (100 mg/kg) after 4 weeks exceeded that the reference drug after 2 weeks. It is evident from the presented results the 70 % methanol total extract (100 mg/kg) possesses significant antihyperglycemic activity followed by the methanol fraction from (100 mg/kg). The least effective was the methanol fraction (50 mg/kg) with percentage of change 14.6% and 37.95% after 2 and 4 weeks, respectively. The 70% methanol total extract (100 mg/kg) of Ipomoea cairica shows a percentage of change in serum glucose level of 17.3 % and 49.8 % respectively. It is evident from the presented results the 70% methanol total extract (100 mg/kg) possess a significant antihyperglycemic activity . The least effective was the methanol fraction (50 mg/kg) with percentage of change 14.2% and 26.9% after 2 and 4 weeks, respectively.

5.3. Antioxidant Activity

The 70% methanol total extract (100 mg/kg) of Jacquemontia pentantha induced highly significant antioxidant activity. The percentage of change is 2.7 % compared to the control healthy group.The least effective was the methanol fraction from the original extract (50 mg/kg), the percentage of change being 19.5%. The 70% methanol total extract (100 mg/kg of Ipomoea cairica induced highly significant antioxidant activity. The percentage of change is 4.9 % compared to the control healthy group. The least effective was the methanol fraction (50 mg/kg), the percentage of change being 22.5 %.

5.4. Anti-inflammatory Activity:

The 70% total methanol extract (100 mg/kg) is the most potent extract as it reduced the edema by 10.1% after 4 hours, this result similar to the reference drug indomethacin (10.1 % change) after 3 hours. The least effective sample was the methanol fraction (50 mg/kg), the percentage of change being 18.1%. The70% methanol total extract (100 mg/kg) of Ipomoea cairica is the most potent as it reduced the edema by 13.7%, after 4 hours.The least effective was the methanol fraction from the original extract (50 mg/kg), the percentage of change being 20.3%.

5.5. Hepatoprotective Activity:

The carbon tetrachloride (CCl4) group shows a significant increase in serum liver enzymes AST, ALT and ALP as compared to control group, indicating liver injury. Treatment of those rats with 70% methanol total extract and methanol fraction from the original extract of aerial part of Jacquemontia pentantha and Ipomoea cairica showed significant decrease in serum AST, ALT and ALP as compared to reference drug (Silymarin). The 70% methanol total extract (100mg) of the tested extracts aerial part of Jacquemontia pentantha has been found to be promising hepatoprotective agent as it reduced the serum liver enzymes AST, ALT and ALP by 29.6, 23.5 and 38.7, respectively, compared to reference drug silymarin (30.1, 25.5 and 59.7), respectively. Next,70% methanol total extract (50 mg/kg) with percentage of change were 23.2%, 22.3% and 26.8%, respectivily . The least effective was the methanol fraction from the original extract (50 mg/kg), the percentage of change being 7.5%, 9.4% and 19.7%, respectively. The 70% methanol total extract(100 mg/kg) of Ipomoea cairica showed significant decrease in serum AST, ALT and ALP by 21.2%, 14.2% and 39.9%, respectively, as compared to reference drug silymarin, followed by methanol fraction (100mg/kg) with percentage of change were 14.7 %, 11.8 % and 28.6 %. The least effective was the methanol fraction from the original extract (50 mg/kg), the percentage of change being 11.3%, 5.6% and 6.8%, respectively.

5.6. Antimicrobial Activity

Results of antibacterial testing of the different concentrations of methanol fraction of Jacquemontia pentantha showed inhibitory activity against the gram positive bacteria B. subtilis and the gram negative bacteria P. aeruginosa with zone of inhibition of 9 mm and minimum inhibitory concentrations (MIC) 62.5 μg. The results also indicated that, all different concentrations of the 70% methanol total extract exhibited a wide range of antibacterial activity against gram positive and gram negative bacteria, with zones of inhibition range from 6 to 9mm. In addition, it showed moderate activity against C. albicans with zone of inhibition of 7 mm, and minimum inhibitory concentrations (MIC) 500 μg .Methanol extract of Ipomoea cairica showed moderate inhibitory effect against the Gram positive bacteria B. subtilis and the Gram negative bacteria P. aeruginosa with zone of inhibition of 9 and 8 mm, respectively , and minimum inhibitory concentrations (MIC) 62.5 and 500 μg . Also exhibited low inhibitory activity against S. aureus, E. coli and C. albicans, with zones of inhibition range from 6 to 7mm. Results also indicated that, 70% methanol total extract exhibited a wide range of antibacterial activity against Gram positive and Gram negative bacteria, with zones of inhibition range from 6 to

10mm. and showed moderate activity against C. albicans with zone of inhibition of 7 mm, and minimum inhibitory concentrations (MIC) 250 μg.

Conclusions:

The study of the phytoconstituents and bioactivities of Jacquemontia pentantha and Ipomoea cairica aerial parts were carried out on 70% methanol total extract and methanol fraction. Isolation, characterization and structure elucidation of the main constituents in the methanol soluble fraction of the aerial parts of Jacquemontia pentantha (Jacq.) G. Don , resulted in the isolation and identification of ten flavonoid compounds were reported for the first time from Jacquemontia pentantha in this work. Quercetin 3-O-ß-D-glucopyranoside (4 g) is a majour compound, while compound Quercetin 3- O-(2-E-caffeoyl-ß-D- xylopyranosyl)–(1→2)–α–L–rhamno-pyranosyl–(1→2)– ß–D– glucopyranosid is isolated for the first time from nature. Evaluation of certain biological activities of Jacquemontia pentantha and Ipomoea cairica aerial parts were carried out on 70% methanol total extract and methanol fraction, showed several activities including acute toxicity (LD50), anti- inflammatory activity, antihyperglycemic activity, antioxidant activity and hepatoprotective activity and also evaluation of

antimicrobial activity of extracts of the aerial parts of both plants . Recommendation based on the results, both plants has low toxicity , this suggests we must direct their application for medicinal purposes and for treatment of human diseases. Cultivation of both plants in reclaimed land in Egypt and studing the stability and bioavailability of their extracts. Applying Good Agriculture Practice (GAP), as organic fertilization, using plant origin pesticides and modern systems of irrigation. Development of phytopharmaceuticals containing the standardized bioactive ingredients.