A Comparative Pharmacognostical Study of

Chrysophyllum oliviforme L. and cainito L. (Family ) grown in Egypt

A Thesis Submitted By

Nariman El-Sayed Hassan Mahdy

For the Degree of Master in Pharmaceutical Sciences (Pharmacognosy)

Under the Supervision of

Prof. Dr. Prof. Dr. Seham Salah El-Din El-Hawary Soheir Mohamed El Zalabani Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Faculty of Pharmacy Cairo University Cairo University

Dr. Nabil Mohamed Aboul-Fotouh Lecturer of Pharmacognosy Faculty of Pharmacy Cairo University

Abstract

A Comparative Pharmacognostical Study of L. and L. (Family Sapotaceae) grown in Egypt

Chrysophyllum oliviforme L. (Satin ) and Chrysophyllum cainito L. (Golden leaf ), two members of the pantropical family Sapotaceae, are successfully acclimatized in Egypt. The current study aimed to evaluate the local as potential candidates for implementation in pharmaceutical industries. Reports concerned with experiment-based bioactivities of the cited were scarce or completely lacking, thus imposing an intensive investigation. To ensure quality and purity of the raw material, criteria for characterization of and/or discrimination between the two species were established via DNA and botanical profiling, proximate analysis and phytochemical screening. The were subjected to comparative biological and chemical study to select the most suitable from the medicinal and economic standpoint. In this respect, the biological activities of the non-polar (petroleum ether) and polar (defatted 70% ethanol) extracts of the tested samples were assessed in-vivo through measuring appropriate biochemical parameters, histopathological examination and genetic study. Meanwhile, the chemical composition of the leaves was examined through qualitative and quantitative comparative analyses of the lipoid and phenolic components. Moreover, the global consumption of the of C. cainito stimulated the nutritional evaluation and flavor characterization of that obtained from the local . Results revealed the safety of all extracts up to 6000 mg/kg b.wt. Besides, the petroleum ether extracts of both leaves were found more efficient as anti-inflammatory and analgesic than their respective ethanol extracts. Yet, the latter were more effective as antihyperglycemic, hepatoprotective and antioxidant. In addition, leaf extracts of C. oliviforme exerted more pronounced biological activities than those of C. cainito. This was referred to their higher lipoid and polyphenol contents, as indicated by yield determination and GC/MS profiling of lipoids, and quantitative colorimetric and HPLC analyses of polyphenols. Leaf extracts of C. oliviforme were selected for intensive chemical investigation aiming to isolate and identify their individual components, based on more pronounced biological activities and apparently higher content of bioactive lipoids and polyphenols. Repeated chromatographic fractionation of the extracts allowed the isolation of twelve compounds. The isolated constituents, identified for the first time from the leaves of C. oliviforme, included three triterpenoids [α-amyrin, β-amyrin and myrianthic acid (2α, 3α, 19α, 23 tetrahydroxy ursenoic acid)], two phytosterols (stigmasterol and stigmasterol-3-O-β-D-glucoside), four flavonoids (quercetin, myricetin, kaempferol and isoquercitrin) and three phenolic acids (caffeic, trans-ferulic and gallic acids). Finally, specification of the nutritional and flavor characteristics of the of C. cainito L. suggested the increase of local propagation of the plant for edible and economical purposes. Key Words: Chrysophyllum oliviforme L., Chrysophyllum cainito L., botanical characteristics, DNA fingerprints, bioactivities, lipoid and phenolic profiling, leaf constituents, fruit nutritive value and aroma.

Introduction

For centuries, herbs have been used to treat many types of ailments. Despite the continuous evolution in synthetic chemistry and biotechnology, plants are still an indispensable source of remedies and modern medicine essentially rely on herbal basis.

Synthetic drugs initially treat harmful symptoms of specific diseases based on scientific pathological examination. Meanwhile, herbal medicines comprising complex mixtures of bioactive constituents, such as phenolics, polysaccharides, mucilage and tannins, may modulate and modify the effects of orthodox medicines to aid the body’s self-healing processes in overcoming any deficiency. Yet, symptom relief constitutes only a section among several efficient therapeutic strategies of medicinal plants. In fact, herbal drugs, made from all-natural ingredients, are much safer and less expensive than synthetic medicines. Thus, products derived from plants and other natural sources (e.g. fungi and marine microorganisms) are expected nowadays to contribute greatly in drug industries (Karimi et al., 2015).

Sapotaceae is a pantropical family of or shrubs, that includes about 70 genera and 800 species and is worldwide distributed in tropical and subtropical regions (Evans, 1998). The family is reputed for its latex (Schan and Schokman, 2004) and for including several species that produce edible fruits like (sapodilla, sapota), Chrysophyllum cainito, and Planchonia careya (Baky et al., 2016).

In addition, other species have been reported to exert a wide range of biological effects such as anticancer, cytotoxic, antioxidant, anti-inflammatory, antiulcer, antimicrobial, antidiabetic, antihyperlipidemic, hypocholesterolemic, hepatoprotective and immunomodulatory activities (Baky et al., 2016).

Chrysophyllum, described as a genus by Linnaeus (Hansen and Maule, 1973), consists of a group of tropical trees belonging to the family Sapotaceae. It comprises about 70-80 species indigenous to northern with one species, C. oliviforme L., extending north to Florida. The attractive colourful upper and lower surfaces of the leaves of the plants of this genus led to their wide propagation as ornamental (Inyama et al., 2016). In folk medicine, Chrysophyllum species are commonly used for treatment of inflammation and diabetes. Moreover, a variety of secondary metabolites such as phenolic acids, flavonoids, triterpenoids, sterols and other lipoids, as well as glycosides, has been reported from these species and certain of its members subjected to biological studies. The latter were mainly concerned with evaluation of their anti-inflammatory, anti-hypersensitive, antioxidant, antidiabetic and antimicrobial effects (Einbond et al., 2004; Luo et al., 2002; Meira et al., 2014; Rasoanaivo et al., 2014).

Chrysophyllum oliviforme L. (Satin leaf) and Chrysophyllum cainito L. (Golden leaf tree) are two members of this genus, which have been successfully acclimatized in Egypt.

The aim of this work was targeted towards assessing the efficacy of these two locally cultivated plants, as a source of potential medicines in order to further increase their propagation.

To fulfil this goal, and following a comprehensive review of the available literature on the genus, a comparative study of the two cited species was planned to include the following:

1. Careful collection and authentication of the plant material.

2. Establishment of useful criteria for taxonomical relatedness and/or discrimination between the two selected species based on genetic and botanical profiling.

3. Evaluation of the biological efficiency of the total non-polar and polar extractives of the leaves of the two plant species through in-vivo studies, namely as anti-inflammatory, analgesic, anti-hyperglycemic, hepatoprotective and antioxidant.

4. Comparative chemical exploration of the leaves of the two plants including proximate analysis, phytochemical screening, examination of successive extractives, and either chromatographic or spectrophotometric analyses of lipoid, phenolic and mucilage components.

5. Selection of the most promising leaf extractives for more intensive chemical investigation guided by the previous biological and chemical studies including isolation, purification and characterization of secondary metabolites with expected medicinal and/or economic value. 6. Investigation of the fruit derived from the locally grown Chrysophyllum cainito L. through determination of its proximate and nutritional composition and analysis of its volatile constituents.

Summary

Genus Chrysophyllum L. (Sapotaceae) comprises 70-80 species of tropical trees. A large number of secondary metabolites including phenolic acids, flavonoids, triterpenoids, sterols and other lipoids, as well as glycosides, have been reported in the genus. Accordingly, Chrysophyllum species were found to exert various activities viz., anti-inflammatory, anti-hypersensitive, antioxidant, antidiabetic and antimicrobial. In folk medicine, the genus is recommended for treatment of inflammation and diabetes. C. oliviforme L. (Satin leaf) and C. cainito L. (Golden leaf tree) are two members of the genus, of worldwide tropical distribution, that have been acclimatized in Egypt. The study is divided into five parts.

Part I: Comparative Genetic and Botanical Profiling of Chrysophyllum oliviforme L. and Chrysophyllum cainito L.

Chapter 1: DNA fingerprinting

The extracted DNA of C. oliviforme L. and C. cainito L. was amplified using 10 primers to reveal RAPD fragments. Each of the 10 primers successfully directed the amplification of a genome-specific fingerprint of DNA fragment; all amplifications were found to be prolific.

Different RAPD fragments were produced by PCR; 104 for C. oliviforme and 112 for C. cainito. The ten primers had produced multiple band profiles with a number of amplified DNA fragments ranging from 14 when S55, S97 and S361 were used in C. oliviforme L. species and 20 fragments being produced by S97 in C. cainito L. species. On the other hand, the least number of fragments was 3, being produced by S83 in C. oliviforme L. species and 4, being produced by S61 in C. cainito L. species. The pattern obtained using S18, S20, S50, S55, S102, S361 and S379 primers was quite identical in the two species. Those primers could, therefore, be used as indicators for obtaining genetic markers. The S61, S83 and S97 primers were found to be the most effective in generating polymorphic bands on application of the RAPD technique to the two Chrysophyllum species. From the previous findings, it could be concluded that the most relevant fragments resulting from the successful combination of templates and primers were those produced by S61, S83 and S97 RAPD primers. Such primers could be used to discriminate between the two investigated species based on the high level of polymorphism between the produced fragments. However, the other estimated RAPD-primers, which showed similarity, could be used in the identification, characterization and authentication of different Chrysophyllum species and accessions.

Chapter 2 and 3: Macro and micro-morphological studies

The botanical features of the different organs of the two plant species were in agreement with the general characters of family Sapotaceae and genus Chrysophyllum L. and could be considered as useful criteria for authentication of the plant material in entire and powdered forms.

Part II: Comparative Biological Investigation of the Leaves of Chrysophyllum oliviforme L. and Chrysophyllum cainito L.

Chapter 1: General procedures and oral acute toxicity study

The petroleum ether and defatted 70% ethanol extracts of the leaves of the two plants were selected for biological evaluation.

The oral acute toxicity study of the extracts revealed that there was no mortality or any signs of behavioral changes or toxicity observed after oral administration of tested extracts up to a dose level of 6000mg/kg body weight in mice.

Chapter 2: Evaluation of the anti-inflammatory and analgesic activities

The anti-inflammatory activity was evaluated in male albino rats by the Carrageenan-induced rat paw edema method in comparison to indomethacin and activity expressed as % inhibition of the induced edema. For evaluation of the central analgesic activity, Eddy's hot plate method was applied using mice and the activity expressed as prolongation of latency period relative to indomethacin.

The peripheral analgesic activity was evaluated in mice by the Writhing method using 1% acetic acid for pain induction. The activity was expressed as the reduction in number of writhes recorded during 15 min relative to ibuprofen.

Results revealed that pretreatment with petroleum ether and defatted ethanolic extracts of the leaves of both plants at both doses (125 and 250 mg/kg body weight) significantly (p<0.05) reduced λ-carrageenan-induced paw edema, increased latency period in Eddy's hot plate method and reduced the number of writhes in writhing method. Those results were confirmed by the evaluation of the effects of tested extracts on haematological parameters.

The petroleum ether extracts of both leaves were found more efficient as anti-inflammatory and analgesic than their respective defatted ethanolic extracts. Moreover, extracts of C. oliviforme exhibited higher activities than those of C. cainito.

Chapter 3: Evaluation of the anti-hyperglycemic, hepatoprotective and antioxidant activities

The extracts showed significant antihyperglycemic activity in STZ-induced diabetic rats with significant improvement in lipid parameters compared to the standard antidiabetic drug Glibenclamide.

The extracts showed significant hepatoprotective and antioxidant activities in CCL4-induced hepatotoxicity in male albino rats with significant improvement in liver and lipid parameters compared to the standard hepatoprotective drug Silymarin.

The defatted ethanolic extracts of both leaves were found more efficient as antihyperglycemic, hepatoprotective and antioxidant than their respective petroleum ether extracts. Moreover, extracts of C. oliviforme exhibited higher activities than those of C. cainito. These results were confirmed by histopathology and genetic studies of the isolated pancreases and livers. Part III: Comparative Phytochemical Investigation of the Leaves of Chrysophyllum oliviforme L. and Chrysophyllum cainito L.

Chapter 1: Proximate analysis, phytochemical screening and extraction

 Quality control criteria of the plants were established through proximate analysis of the leaves. These included determination of total ash (3.65 & 5.66 g %), acid-insoluble ash (0.9 & 1.58 g %), water-insoluble ash (1.18 & 1.42 g %) values, crude fiber (12.4 &10.42 g %) and moisture (53.04 & 51.97 g %) contents.

 Preliminary phytochemical screening of the leaves revealed the presence of carbohydrates and/or glycosides, flavonoids, sterols and/or triterpenes and tannins in both samples.

 The air-dried powdered leaves of Chrysophyllum oliviforme L. and Chrysophyllum cainito L. (2 Kg, each) were, separately, subjected to exhaustive cold maceration. The samples were first extracted with petroleum ether (60-80oC), followed by extraction of the defatted dry marc with ethanol 70% and the ethanol-free extractive suspended in water, and fractionated by shaking with methylene chloride, ethyl acetate and n-butanol saturated with water, in succession.

 The percentage yield of the different extractives was the highest for petroleum ether followed by ethyl acetate, methylene chloride then n-butanol. The yield of Chrysophyllum oliviforme L. extractives was higher than those of Chrysophyllum cainito L. except for the n-butanol ones which were nearly similar.

 The preliminary phytochemical screening revealed that the ethanol (70%) extracts gave strong positive tests for carbohydrates and/or glycosides, flavonoids, sterols and/or triterpenes; and a weaker response to those of tannins and saponins. Sterols and/or triterpenes appeared to be the main components of the petroleum ether and methylene chloride fractions. Carbohydrates and/or glycosides and polyphenols were detected in both the ethyl acetate and n-butanol fractions; whereas, saponins were traced in the n-butanol fraction only.

Chapter 2: Analysis of phenolic components

I. Spectrophotometric analysis

Total phenolic and flavonoid contents were determined in the defatted ethanolic extracts of the leaves by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. A Shimadzu spectrophotometer (UV-1650PC) was used.

 For total phenolic content: the absorbance of the blue color was measured at 765nm. Gallic acid was used as standard and equivalents determined (w/w) from a pre-established concentration curve (y = 0.0041x - 0.016, R² = 1). The absorbance recorded for C. oliviforme extract was (1.524±0.0015) vs (1.082±0.0031) for C. cainito corresponding to total phenolic content of (375.66±0.37) vs (267.69±0.92) μg gallic acid equivalent / mg ethanolic extracts.

 For total flavonoid content: the absorbance of the yellow color was measured at 415nm. Quercetin was used as standard and equivalents determined (w/w) from a pre-established concentration curve (y = 0.0094x + 0.0181, R² =0.9964). The absorbance recorded for C. oliviforme extract was (0.121± 0.00058) vs (0.549±0.001) for C. cainito corresponding to total flavonoid content (10.98±0.061) vs (56.47±0.001) μg quercetin equivalent / mg ethanolic extracts.

 Polyphenol contents were higher in C. oliviforme than in C. cainito; in contrast, the latter was richer in flavonoids.

II. High performance liquid chromatographic analysis

HPLC fingerprinting was conducted to investigate the degree of similarities and/or differences among the phenolic profiles of the 2 plants .The experimental results revealed that the concentrations of total identified phenolic compounds were 1293.27±4.1 and 59.86±8.38 mg/100g respectively, of the dried extracts of the leaves of C. oliviforme and C. cainito. Sinapic acid and gallic acid were the major identified phenolic compounds with concentrations; 589.47±0.21 and 32.92±2.25 mg/100g respectively, in the dried ethanol extracts of the investigated leaves. Chapter 3: Analysis of lipoids and mucilage

I. Gas Chromatography/Mass Spectrometry (GC/MS) analysis of lipoids

 The unsaponifiable and saponifiable lipoids were prepared from one gram of the petroleum ether extracts of the leaves of C. oliviforme L. and C. cainito L. The yields of the unsaponifiable and saponifiable matters were 0.66 vs 0.6 g and 0.3 vs 0.21 g, respectively.

 Constituents identified by GC/MS analysis of the unsaponifiable fractions of the petroleum ether extracts of C. oliviforme & C. cainito represented 93.89 vs 76.6% of the total composition. Sterols & triterpenes predominated (71.11 vs 60.93%); among these α-amyrin (42 vs 44.69%) & β-amyrin (27.05 vs 14.81%) were major. Beside, unusual aromatic hydrocarbons were detected in both samples.

 Constituents identified by GC/MS analysis of the fatty acid methyl esters in the saponifiable fractions of the petroleum ether extracts represented 91.72 vs 82.33% of the total composition, with 9, 12, 15 octadecatrienoic acid (z, z, z) prevalent in both samples (27.21 vs 35.77%)

II. High Performance Liquid Chromatographic (HPLC) analysis of mucilage hydrolysates

 Sugars identified by HPLC represented 91.26 and 97.99 %, respectively of the total composition of the mucilage hydrolysates of the leaves of the two plants.

 A total number of 9 sugar components were identified in the examined samples, under the adopted operating conditions, among which 5 were common in the 2 samples.

 Uronic acids were detected in variable amounts with glucuronic acid prevailing the composition of C, oliviforme mucilage hydrolysate, and galacturonic acid, although not detected in this sample, was identified in an appreciable amount (23.60%) in that of C. cainito.  Whereas, the major detected monosaccharides were glucose (27.91%) and xylose (5.59%) in the C. oliviforme sample and fructose (54.79%) and galactose (16.95%) in that of C. cainito.

Part IV: Isolation and identification of the major constituents of the leaves of C. oliviforme L.

The biological investigation revealed that, compared to Chrysophyllum cainito L., the non-polar petroleum ether extract of the leaves of C. oliviforme L. was found more efficient as anti- inflammatory and analgesic (pages 120 - 131), besides being obtained in a better yield (page 197). Moreover, its polar ethanol (70%) extractives exerted more pronounced antihyperglycemic, hepatoprotextective and antioxidant effects relative to those of the leaves of C. cainito L. (pages 138 – 189), alongside being acquired in a higher amount (page 197).

Taking all the previous facts in consideration, the extracts of the leaves of C. oliviforme were selected for further intensive chemical investigation aiming to isolate and identify their component bioactive metabolites.

Chapter 1: Isolation and identification of the constituents of the petroleum ether extract

 The petroleum ether extract of the leaves of C. oliviforme was chromatographed on a vacuum liquid chromatography column and the produced subfractions monitored by TLC. Those giving a strong response to the presence of sterols and/or triterpenes were rechromatographed on different silica gel columns. This process led to the isolation of three compounds which were subjected to 1H-NMR and 13C-NMR spectral analysis in addition to comparison to published data. The isolated compounds were identified as two pentacyclic triterpenoid

compounds (P1, β-amyrin and P2, α-amyrin) and a phytosterol (P3, stigmasterol).

 As far as the available literature is concerned, this is the first report on isolation of all three compounds from the leaves of Chrysophyllum oliviforme L. Although α- and β-amyrins have been reported earlier from other Chrysophyllum species viz., C. flexuosum Mart. and C. marginatum Hook. & Arn as indicated in table 1 (page 7). Meanwhile, stigmasterol was previously isolated from C. albidum G. (Idowu et al., 2016).

 In-vitro and in-vivo biological studies on α- and β-amyrins revealed that these compounds exert significant pharmacological effects against various health-related ailments including; inflammation, alongside microbial, fungal and viral infections (Vázquez et al., 2012). The bioactive efficiency of stigmasterol has also been established especially as thyroid inhibitory, antiperoxidative and hypoglycemic (Panda et al., 2009).

 Therefore, the potential anti-inflammatory and analgesic activity of the petroleum ether extract of the leaves of C. oliviforme L. (pages 120 - 131), might in part be attributed to the presence of amyrins and stigmasterol, especially that these compounds constitute the bulk of its unsaponifiable fraction, as previously established (page 213).

Chapter 2: Isolation and identification of the constituents of the methylene chloride fraction

 The methylene chloride extract was studied to investigate the nature of its major constituents suggesting that they might contribute to the biological activities of the parent ethanolic extract. In this respect it was subjected to VLC and coupled to TLC monitoring. Subfractions that appeared enriched in sterols and/or triterpenes were rechromatographed on different silica gel columns and resulted in isolation of two compounds which were subjected to 1H- NMR and 13C-NMR spectral analysis in addition to comparison to published data. These compounds were identified as: myrianthic acid (2α, 3α, 19α, 23 tetrahydroxy urs-12-en-28-

oic acid, Compound M1) alongside stigmasterol 3-O-β-D-glucoside (Compound M2).

 As far as the available literature is concerned, this is the first report on isolation of both myrianthic acid and stigmasterol 3-O-β-D-glucoside from the genus Chrysophyllum L.

 Among natural triterpenoids, ursolic acid and its derivatives are reputed to exhibit a vast array of pharmacological activities. Besides their established anti-cancer effect, these compounds were claimed as protective to lungs, kidneys, liver and brain as well as anti- inflammatory, antimicrobial, antiviral and antiprotozoal. Their anabolic effects on skeletal muscles and ability to suppress bone density loss leading to osteoporosis were also recorded (Woźniak et al., 2015). Likewise, stigmasterol has been proven to exhibit a thyroid inhibitory effect, alongside antiperoxidative and hypoglycemic activities (Panda et al., 2009).

 These compounds might, therefore, contribute to the previously determined anthyperglycemic, hepatoprotective and antioxidant potential of the parent ethanol extract of the defatted leaves of Chrysophyllum oliviforme L. (pages 138 – 189).

Chapter 3: Isolation and identification of the constituents of the ethyl acetate fraction

 The composition of the polar fractions of the ethanol extract mainly its ethyl acetate fraction might be responsible for its anti-diabetic, hepatoprotective and antioxidant activities were also investigated. Therfore, this extract was subjected to different chromatographic techniques aiming to isolate and identify its major constituents.

 The ethyl acetate fraction was thus subjected to VLC and TLC monitoring. Subfractions which appeared riched in phenolics were rechromatoraphed on different sephadex LH-20 columns. Thus resulted in isolation and identification of four flavonoids and three phenolic acids. The isolated compounds were identified on the basis of their physicochemical and UV, 1H-NMR and 13C-NMR spectral data and included:

 Four flavonol derivatives among which 3 aglycones viz., kaempferol (Compound E1),

quercetin (Compound E2) and myricetin (Compound E3); in addition to a glucoside,

isoquercitrin (Compound E7).

 Three phenolic acids namely, trans-ferulic (Compound E4), caffeic (Compound E5) and

gallic acids (Compound E6).

 As far as the available literature is concerned, this is the first report on isolation of these seven compounds from the leaves of Chrysophyllum oliviforme L. Although being reported from other members of the genus viz., C. albidum G., C. cainito L., C. flexuosum Mart. and C. marginatum Hook. & Arn., as recorded in tables 2 and 3 (pages 11 and 14).

 The majority of these phenolics have been reported to exert potent antioxidant activities (Koffi et al., 2009), and therefore are expected to contribute efficiently to the observed anthyperglycemic, hepatoprotective and antioxidant effects of the total ethanol (70%) of the defatted leaves (pages 138 – 189).

Part V: Phytochemical Investigation of the Pericarp of the Fruit of Chrysophyllum cainito L.

Chapter 1: Proximate analysis and nutritional evaluation of the pericarps

 Proximate analysis of the leaves was carried out adopting the method of the Association of Official Analytical Chemists (AOAC 2000) where the results showed a total ash value of (0.79 g %), acid-insoluble ash value of (0.2 g %), water-insoluble ash value of (0.5 g %), crude fiber (5 g %) and moisture content value of (77.46 g %).

 Total protein, lipid and carbohydrates, amino acid composition, vitamins and mineral contents were analyzed.

 The high moisture content and considerable amount of fibers and carbohydrates besides the low fat content support the use of the analyzed pericarp in controlled calorie diets with optimal nutritional value.

 The essential amino acid lysine was the major identified component. Meanwhile, proline was the amino acid which was detected in the lowest amount. Besides, the pericarp of C. cainito L. was found to have low vitamins B contents although being rich in vitamin C. In addition, the pericarp of C. cainito L. was rich in mineral elements. The highest element was phosphorus followed by potassium and the lowest element was iron. Chapter 2: Effect of extraction techniques on the volatile constituents of the pericarp

 A qualitative and quantitative variability was observed in the composition of the volatiles of the pericarp prepared by the two extraction techniques adopted in this study. The relative decrease observed in some of the major hydro-distilled components in the HS/GC-MS profile might be attributed to the identification of a large number of minor components in the latter.

 In fact, HS/GC-MS allowed the identification and quantification of 37 components that represented 97.87% of the total composition. Meanwhile, only 14 components out of 17 were identified in the hydro-distilled sample representing 96.65% of the total composition.

 Although alcohols were identified as major components in both samples, yet their relative amount was much higher in the hydro-distilled sample (91.32%) than in the HS extracted one (52.66%).

 Whereas, a relatively large amount of hydrocarbons 25.91% was identified in the HS extacted sample; yet, these compounds could not be detected in the hydro-distilled one.

 The significant improvement in the number of identified components observed when using HS/GC-MS confirms that this technique is more precise, reliable and faster for investigation of the volatiles in the least amount of analyzed samples. Moreover the different GC-MS operating conditions adopted in the two procedures might affect the resulting chromatographic profiles of the analyzed samples.

 By comparing the results of the two used techniques with the published data concerning the composition of the volatiles of the pericarps (Pino et al., 2002); in which terpenoids comprised the largest class of volatiles (36.4%) and (E)-2-hexenal, 1-hexanol, limonene, linalool, α-copaene and hexadecanoic acid were found to be the major constituents. The qualitative and quantitative variations were observed in the chromatographic profiles, may be attributed to climatic and/or geographical factors.

General Conclusion

1. DNA fingerprinting allowed discrimination between Chrysophyllum oliviforme L. and C. cainito L.

2. The botanical features of the different organs of the two plant species were in agreement with the general characters of family Sapotaceae and genus Chrysophyllum L. and could be considered as useful criteria for authentication of the plant material in entire and powdered forms.

3. The petroleum ether extracts of the leaves of the two plants were found more efficient as anti-inflammatory and analgesic than their respective defatted ethanolic extracts. Yet, the latter were more effective as antihyperglycemic, hepatoprotective and antioxidant.

4. Leaf extracts of C. oliviforme exerted more pronounced biological activities than those of C. cainito. This might be attributed to their higher lipoid and polyphenol contents, as indicated by the GC/MS profiles of lipoidal matters, as well as data recorded during colorimetric and HPLC analyses of phenolics and flavonoids. Accordingly, these extracts were selected for more intensive phytochemical investigation.

5. Twelve compounds were isolated and identified from the different extracts of the leaves of C. oliviforme for the first time including:

 Three triterpenoids [α-amyrin, β-amyrin and myrianthic acid (2α, 3α, 19α, 23 tetrahydroxy ursenoic acid)].  Two phytosterols (stigmasterol and stigmasterol-3-O-β-D-glucoside)  Four flavonoids (quercetin, myricetin, kaempferol and isoquercitrin)  Three phenolic acids (caffeic, trans-ferulic and gallic acids).

6. Data of proximate analysis, nutritional evaluation and GC/MS investigation of the volatiles of the fruits of C. cainito L. were determined. The headspace technique was found more reliable and faster than hydro-distillation for extraction of the fruit volatiles as indicated by the GC/MS profiles. Specification of the fruit characteristics suggested the increase of local propagation of the plant for edible and economical purposes. Recommendations

Nowadays, the demand for herbal medicine is continuously increasing due to the serious side effects of synthetic drugs.

In this respect, the leaves of Chrysophyllllum oliviforme L. and Chrysophyllum cainito L. might be recommended as valuable nutritional supplements for prevention and/or control of various ailments like Alzheimer's disease, hyperglycemia, pain, as well as hepatotoxic and inflammatory disorders.

However, extensive clinical trials must be performed in order to support all the evaluated bioactivities and to facilitate implementation of these promising herbals in pharmaceutical formulations.