Chemical Profile and Biological Activity of Casimiroa Edulis Non-Edible Fruit`s Parts

Purpose: the non-edible fruit parts of Casimiroa edulis Llave et were evaluated for their active constituents and their potential as antioxidants, anti-inflammatory and antitumor activity. Methods: Fruits peel (FP) and seeds kernel (SK) of Casimiroa edulis Llave et Lex. were extracted successively with hexane and then methanol. Fatty acids were prepared from hexane extracts and identified by GC. Total flavonoid, phenolic acids and tannins contents in methanol extracts were determined by UV spectrophotometer and identified by HPLC. Antioxidant, in-vitro anti-inflammatory activity and antitumor effect against Caco-2 cell line were determined. Results: GC analysis of hexane extracts showed that oleic acid (47.00%) was the major unsaturated fatty acids in both extracts while lignoceric acid (15.49%) is the most abundant saturated fatty acid in (FP). Total phenolic, flavonoid and tannin contents in (FP) & (SK) methanol extracts were; 37.5±1.5, 10.79±0.66 and 22.28±0.23 for (FP); 53.5±1.5mg/g, 14.44±0.32 mg/g; and 53.73±3.58 mg/g for (SK) respectively. HPLC analysis of methanol extract revealed that; the major phenolic compound was pyrogallol in (FP) and p-hydroxybenzoic acid in (SK), the major flavonoid was luteolin 6-arabinose-8-glucose in (FP) and acacetin in (SK). Conclusion: This study showed that non-edible parts of C. edulis fruit is a rich source of different phenolic compounds and fatty acids which has great antioxidant, anti-inflammatory and antitumor activities; that could be used as a natural source in pharmaceutical industry.


Introduction
Family Rutaceae; is a small family made up of cultivated fruiting trees and medicinal herbs frequently called citrus family, it has a great economic importance because of its several edible Citrus fruits as orange, lemon, etc. Family Rutaceae is dispersed all over the world, particularly in warm climate and tropical areas, mostly found in Africa and Australia. 1 Casimiroa edulis Llaveet Lex. is a non-citrus fruit belongs to this family, it is commonly known as Zapote blanco or white sapota and mainly cultivated in Mexico and Central America. C. edulis is widely consumed in different parts of the world for its valuable fruit; 2 as it is a rich source of sugar, protein, ascorbic acid, phenols, carotenoids, polyunsaturated fatty acids and minerals like Fe, Cu, Zn, Ca and K. 3 It is traditionally used in many countries as a sleep inducer as it has interesting sedative-like effects. 2 C. edulis leaves and seeds were found to affect blood pressure, cardiac activity aortic muscular tone, 4 and to possess anticonvulsant activity. 5 Methanol extract of C. edulis leaves also showed strong antioxidant activity. 3 Different classes of compounds were previously separated from different parts of C. edulis; furocoumarins and polymethoxyflavones were isolated from the leaves that exhibited adipogenesis activity. 6 Moreover the leaves essential oil had promising antimicrobial activity and mainly contain sesquiterpene hydrocarbons as major constituents. 7 Zapotin; a flavanoidal compound which considered as chemo-preventive agent was isolated from the seeds; it was also chemically synthesized because of its great anticancer activity. 8 Different compounds were also isolated from the seeds methanolic extract and showed great cardiovascular activity. 9 Several studies reported that; C. edulis can be considered as valuable plant, so the aim of this study is to evaluate the importance of the non-edible parts of C. edulis fruit to evaluate its chemical composition as well as antioxidant, anti-inflammatory and antitumor potential.
Preparation of plant extract 100 gm fruit peel (FP) and 100 gm seed kernel (SK) of C. edulis were separately coarsely powdered and extracted with n-hexane then by methanol for 72 h using a Soxhlet extractor at 60°C. All the extracts were dried separately under reduced pressure.

Chemical composition GC analysis of the Fatty Acids composition of hexane extract
Hexane extracts of (FP)He and (SK)He were subjected separately to direct methylation in 1.5% sulfuric acidmethanol at 95°C for 2 h. 10 Total Flavonoid, Phenolic acids &Tannins content in methanol extract of C. edulis This was determined for the methanol extracts of (FP)Me and (SK)Me according to methods described previously. 11,12 HPLC Analysis of the methanol extracts The phenolic and flavonoid compounds of (FP)Me and (SK)Me of C. edulis were extracted according to the method described by Mattila et al. 13 Biological activity for methanol and hexane extracts Antioxidant activity of C. edulis extracts using ABTS, DPPH and Total antioxidant activity It was carried out according to Arnao et al., 14 Ye et al. method. 15,16 In vitro Antitumor activity The activity was tested on Caco-2 cell line using sulforhodamin B assay. 17 In vitro Anti-inflammatory activity using bovine albumin serum This was tested using the method of Rahman et al. 18

Statistical analysis
All result is expressed as mean value of three replicate. Data were statistically analyzed through analysis of variance (ANOVA) and Duncans test at P>0.01 using CoStat Statistics Software.

Chemical composition
Fatty acids composition of C. edulis hexane extracts " Table 1" showed that Both (FP)He and (SK)He extracts revealed high percentage of total unsaturated fatty acids 71.15% and 94.20% respectively. The monounsaturated fatty acids oleic acid (omega-9) is the most abundant in both extracts; (36%) in (FP)He and (47%) in (SK)He; Also palmitoleic acid was found in (FP)He (20%) and (SK)He (21%). Furthermore, the hexane extracts showed the presence of different long chain mono and poly unsaturated fatty acids. The unsaturated fatty acids have a great role in decreasing the risk of certain cancers, as colon cancers, breast and prostate. 19 (FP)He has higher percent of total saturated fatty acid 28.85% than that in (SK)He 5.8%; lignoceric acid 15.49% was the major in (FP)He while palmitic acid 3.01% was the highest in (SK)He; these fatty acids play important role in increasing LDL cholesterol level. 20

Biological activity Antioxidant activity of C. edulis extracts
The antioxidant activity of the methanol and hexane extracts of both (FP) and (SK) was evaluated using the ABTS and DPPH free radical-scavenging assay; " Figure 1a and 1b" showed that the (SK) extracts has higher antioxidant activity than the (FP) extracts, this may be attributed to the higher unsaturated fatty acid, phenolic & flavonoid contents. On the other hand the results showed that most powerful antioxidant activity is presented in the (SK)He extract " Figure 1c"; this could be due to the high percentage of the unsaturated fatty acids 94.2% " Table 1" especially oleic acid which has great role in protection of cell membranes from free radicals. 21 The antioxidant activity was also previously reported in the edible parts and leaves methanol extract of C. edulis. 3 In vitro Anti-inflammatory activity Results in " Figure 2" showed that the (SK)He extract at different doses (50, 100 and 150µg/ml) has the most potent anti-inflammatory activity compared with (Diclofenac) as control drug. This effect may be due to the high percentage of the unsaturated fatty acids in (SK)He extract; 22 the potential anti-inflammatory activity of the methanol extracts can be also attributed to the presence of higher percentage of phenolic contents. Antitumor activity of C. edulis extracts " Figure 3" reveled that (FP) and (SK) extracts have certain inhibition effect against the Caco-2 cell line but the most active extract is the (FP)He extract when compared with reference drug doxorubicin, where the IC50 is 45 µg/ml. Conclusion C. edulis non edible fruit parts could be considered as a valuable source for different useful metabolites as unsaturated fatty acid in the hexane extract and polyphenolic, flavonoids and tannins in methanol extract; both extracts revealed great importance as antioxidant, anticancer and anti-inflammatory activities. Thus the nonedible part of fruit which is considered as waste product may be phyto-therapeutically used. However; further in vivo studies are required to authenticate such biological activities in order to formulate safe effective pharmaceutical herbal product.

Ethical Issues
Methods are done after approval of the research ethics committee; the approval form has serial no. REC-FPSPI-5/34.