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Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 Impact Factor (GIF): 0.615 Impact Factor (SJIF): 2.092 June-August 2016; 4(2):142-152

Original Research Article

Chromatographic Study of the Ethanol Extract of myxa L. Fruit Tarik Z. T. Shwaish1*, Faris J. M. Al-Imarah 2

1Gifted Students’ School of Basra, Basra, Iraq 2Dept. Chem. & Marine Environ. Poll., Marine Science Center, University of Basrah, Basra, Iraq

*Corresponding Author: Tarik Z. T. Shwaish, 61001, Gifted students’ school, Arabian Gulf district, Basra, Iraq Received: 15 May 2016 Revised: 25 May 2016 Accepted: 30 May 2016 ABSTRACT

The aim of this study was to perform possible phytochemical screening for the Cordia myxa L. fruit extract by some analytical techniques. Ethanol extract was obtained by treating dried fruit sample with

ethanol, the extract was used for GC and the peaks were studied to determine the composition and volatility of the extract. The extract was also analyzed by using HPLC and further analysis was carried out to determine the presence, concentration, and carcinogenic effects of 16 polycyclic aromatic hydrocarbons. The GC analysis revealed the presence of 10 alkaloids with one major component comprising about 64% of the total extract. In addition, 16 compounds were also reported with HPLC analysis. The presence of the following 6 PAHs in the extract was confirmed: Flourene, Pyrene (B2 carcinogen), Chrysene (B2), Phenanthrene (A1), Flouranthene (A1), and Benzo[a]anthracene with total concentration of about 412 ng/g. From the results, we can conclude the presence of many alkaloids and we can predict that most of the medical effects of the fruits are in the major compound which needs more standardization for further identification. The second finding of the study is the probable carcinogenic and toxic effects of the fruit due to the presence of several PAHs.

Keyword: GC; HPLC; chromatography; ethanol extract; extract; Cordia myxa; PAHs; phytochemistry;

toxicity

INTRODUCTION are used medicinally in different The subject of phytochemistry or plant countries, and they are the source of many chemistry, has developed to be a distinct potent and powerful drugs. Plants have been an discipline somewhere in between natural important source of medicine with qualities for product chemistry and plant biochemistry. thousands of years. [1] Phytochemistry is concerned with the enormous

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variety of organic structures that are elaborated Gastroprotective [6], antioxidant and analgesic and accumulated by plants and deals with the effects [23]. In traditional medicine, Cordia myxa chemical structure of these substances, their L. fruit is used as diuretic, demulcent and in the biosynthesis, turnover, and metabolism, their treatment of stomach aches, coughs and chest natural distribution and their biological function complaints [7]. The fruit is also used externally as [2]. an emollient plaster to maturate abscesses, to Cordia myxa is a species of in the calm rheumatic pain and as an anti-parasitic on family, . It is a medium- ringworm. In addition, the sticky, mucilaginous sized broad-leaved deciduous tree. Common pulp of the fruit is the source of a well-known names include Lasura, Assyrian Plum, Pidar, medicine in the near and middle east called Panugeri, Naruvilli, Geduri, Spistan, Burgund “sapistan” which is useful in the treatment of dulu wanan, Ntege, and Bumber [3] [4]. The coughs, sore throats and chest-complaints on Cordia myxa plant parts are widely used in the account of its demulcent property [8]. traditional medicine especially in the Middle The fruit of Cordia myxa L. has been determined East, India, and China. The seeds, roots, leaves, to have the probability of the presence of some and other parts of the plant have various uses in Pyrrolizidine alkaloids [9]. Moreover, prior traditional medicine [5]. studies have confirmed the presence of the non- The fruit begins appearing in July-August. It is toxic pyrrolizidine alkaloid macrophylline but no light pale to brown in color and it to tends to actual evidence of the concentration of these darken when ripening proceeds. Being full of alkaloids has been reported [24]. viscid glue like mucilage, the pulp is somewhat Concentrated ethanol was chosen for this study translucent. When fully ripe the pulp becomes because it has been widely used as a solvent in quite sweet in taste. [3] The origin of this species pharmacology and phytochemical science and is the region stretching from the eastern has been proved to be the best solvent to extract Mediterranean to eastern India. It has been the maximum components from a plant sample introduced long ago to tropical Africa, tropical compared with other solvents that might be Asia, and Australia, and more recently to the suitable for the extraction process [10] [11] [12] Americas [3] [4]. (Table 1). Cordia myxa plant has many medical uses, it has been long studied for its anti-inflammatory [5],

Table 1: Phytochemical screening in various plant extracts [6] Phytochemicals Petroleum ether Chloroform Acetate Ethanol Flavonoids - - + + Alkaloids - - - + Tannins - - + + Glycosides + + + + Steroids + + + + Phenols - - + + Cardiac glycosides + + + + Saponins + + + + Carbohydrates + + + + Proteins - - - + ‘+’ Presence, ‘-’ Absence

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min. isothermal) to 280oC (30 min isothermal). MATERIALS AND METHODS The total running time was 33.3 min. This study consists of three main stages: Ethanol The HPLC test was run by using Shimadzu UFLT extraction of some bioactive components in the equipped with a gradient pump and manual fruit, Gas chromatography for the determination injector. The separation column had internal of the volatility of the extract as well as some low diameter of 4.6 mm and total length of 25 molecular weight alkaloids, and HPLC for the meters, the bonded phase was Carbon-18 with determination of some compounds found in the pore size of 5-100 μm. The separation mode was ethanol extract and measuring their relative normal phase. The detector type was UV-Visible concentrations [13] [14]. spectrometer. The ambient temperature was o Cordia myxa totally dried black whole fruit 25 C. Shimadzu LC solution computer software sample was cultivated and shade-dried in Basra was used for control and analysis. southern Iraq during summer. The fruit sample was identified by morphological comparison RESULTS with traits described by AP database provided by The results of gas chromatography test Conservatory and Botanical Garden/ Geneva and described earlier were as shown in the gas South African National Biodiversity Institute chromatogram shown in fig. 1 and 65 peaks of [15]. The totally dried fruit sample was grinded different compounds were identified and into fine powder alongside with the seeds by described in terms of peak number, retention using Philips HL7720 mechanical grinder. time, area, height and concentration in table 2. The required amount of fruit was weighed and The chromatogram was very crowded in the was fully immersed with ethanol absolute of retention time in the range of 5-10. The 99.8% concentration at room temperature. The chromatogram was optimized and the modified extract was shaken each hour for the first 12 major peaks were as shown in fig. 2. The solvent hours and was then left for another 12 hours, the peak was removed and the relative composition process was repeated for the next 72 hours. The of the sample was obtained alongside the extract was dried over anhydrous sodium sulfate description of the modified peaks as shown in powder and was then filtered and Nitrogen gas table 3. was passed over the extract for degasification The results of the HPLC test described earlier for purpose. the ethanol extract were as shown in the The extract was diluted and directly employed chromatogram in fig. 3 and the peaks were for gas chromatography. The GC test was run characterized as in table 4 in terms of retention using Shimadzu 2014 gas chromatography time, height, area, and relative composition system coupled with FID detector equipped with shown as percentage of height and area (area is silica wax polar standard capillary tube with considered to be more accurate). length of 59 meters and internal diameter of The presence and concentration of 16 poisonous 0.25 μm. Nitrogen gas (99.999%) was used as the and carcinogenic polycyclic aromatic carrier gas with flow rate of 4 ml/min and an hydrocarbons (PAHs) was obtained from the injection volume of 2 μl was employed manually results of HPLC analysis in nanogram per gram of in the system; injector temp. was 290oC. The sample with total concentration of 411.5 ng/g as oven temp. was programmed from 40oC (0.5 shown in table 5.

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Table 2: Gas chromatography peaks description Peak # Ret. Time Area Height 1 5.280 3498 650 2 5.494 1524 266 3 5.655 32899 5600 4 5.955 4938 2287 5 6.098 11115522 1665234 6 6.206 5012583 1639231 7 6.254 5043586 1644037 8 6.301 4659237 1676138 9 6.452 16893119 1764980 10 6.498 5382162 1756027 11 6.548 4381933 1744094 12 6.604 585212 1753413 13 6.646 5796895 1738379 14 7.311 195091024 6585856 15 7.488 75745170 6925636 16 7.536 23350365 6982197 17 7.601 25457179 7028199 18 7.645 23492366 7041280 19 7.702 21502692 7023398 20 7.750 117692344 7020179 21 8.039 28875391 6493918 22 8.122 21406896 6398641 23 8.175 17845731 6395576 24 8.223 19779711 6443753 25 8.413 69710457 6683419 26 8.456 20620660 6715330 27 8.485 9386751 6716311 28 8.532 18753324 6714604 29 8.577 22392439 6709636 30 8.624 18588923 6675126 31 8.670 13009825 6656951 32 8.714 72244199 6664303 33 13.668 1251 125 34 14.711 3416 346 35 14.747 2365 310 36 17.482 8775 578 37 17.976 1547 389 38 18.182 1671 236 39 18.298 3865 248 40 18.532 1706 309 41 18.630 3583 260 42 19.829 2941 95 43 20.575 183446 11263 44 20.623 25069 11232 45 20.678 49742 11157 46 20.736 24604 11001 47 20.800 66480 10854 48 20.879 76000 10593 49 21.000 70054 10191 50 21.103 55908 9748 51 21.211 152529 9193 52 21.504 25516 7721

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53 21.561 312967 7436 54 25.970 35109 1592 55 26.143 2028 1059 56 26.227 7235 792 57 26.363 1219 312 58 27.665 11117 793 59 27.782 4973 832 60 27.875 13009 810 61 28.220 5870 474 62 33.041 14636 725 63 33.104 4080 684 64 33.213 1666 494 65 33.269 1357 409 Total 880300389 143387010

Table 3: Peaks of the optimized GC chromatogram

Peak # Ret. Time Area Height Conc. Relative conc. 1 7.645 878551085 7039409 99.823 (removed) 2 15.712 113914 3551 0.013 7.345 3 17.976 94882 1489 0.011 6.215 4 20.623 1000004 10747 0.114 64.407 5 25.970 126870 2460 0.014 7.910 6 27.782 35659 822 0.004 2.260 7 29.843 19174 335 0.002 1.130 8 32.024 7266 287 0.001 0.565 9 33.104 130433 1246 0.015 8.475 10 38.962 23794 6 0.003 1.695 11 40.740 3827 -0 0.000 0.000 Total 880106908 7060352

Table 4: Peaks of the HPLC test

Peak # Ret. Time Area Height Area % Height % 1 5.371 5172441 688474 8.008 13.537 2 5.525 4288459 549570 6.639 10.805 3 5.731 1133264 135526 1.745 2.665 4 6.000 5283495 525671 8.179 10.336 5 6.928 46405464 2980535 71.841 58.602 6 7.734 55221 8349 0.085 0.164 7 8.415 56791 6888 0.088 0.135 8 8.827 220861 22827 0.342 0.449 9 9.206 467024 35183 0.723 0.692 10 9.615 20785 2933 0.032 0.058 11 9.903 291966 27295 0.452 0.537 12 10.185 4239 788 0.007 0.015 13 10.497 217890 25656 0.337 0.504 14 10.630 442400 32777 0.685 0.644 15 10.995 179173 14338 0.277 0.282 16 11.231 220251 15766 0.341 0.310 17 11.468 134712 13456 0.209 0.265 Total 64594434 5086033 100.000 100.000

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Table 5: The concentration of 16 PAHs determined by HPLC analysis

No. Compound Name Concentration in ng/g 1 Naphthalene --- 2 Acenaphythylene --- 3 Acenaphthene --- 4 Flourene 187.75 5 Phenanthrene 23.96 6 Anthracene --- 7 Flouranthene 22.08 8 Pyrene 144.77 9 Benzo[a]anthracene 2.71 10 Chrysene 30.28 11 Benzo[b]Flouranthene --- 12 Benzo[k]Flouranthene --- 13 Benzo[a]Pyrene --- 14 Dibenzo[a,h]anthracene --- 15 Benzo[g,h,l]perylene 16 Indeno[1,2,3-cd]Pyrene --- Total 411.55

Table 6: Carcinogenic and other health effects of the identified PAHs

No. Compound Name Structural formula Health effects 1 Flourene 1. Not classifiable as human carcinogen. No data are available in humans. Inadequate evidence of carcinogenicity in animals; D [16]. 2 Pyrene 1. Probable human carcinogen (lung, kidney, and skin cancer). No human data and insufficient data from animal bioassays; B2 [17]. 2. Dermatitis and bronchitis.[18] 3 Chrysene 1. Probable human carcinogen. No human data and sufficient data from animal bioassays; B2. 2. Dermatitis and bronchitis [19].

4 Phenanthrene 1. Confirmed human carcinogen; A1. 2. Irritation of skin and respiratory tract; cough, sore throat and dermatitis [20]. 5 Flouranthene 1. Confirmed human carcinogen; A1 [21].

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6 Benzo[a]anthracene 1. Suspected human carcinogen; A2 [22].

Fig. 1: Chromatogram for the gas chromatography test

Fig. 2: Optimized chromatogram for the gas chromatography test

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Fig. 3: Chromatogram for the HPLC test

DISCUSSION obtain the Kovats retention index of the major The gas chromatogram shown in fig. 1 peak (retention time 20.6) which accounts for characterized around 65 peaks and the test was the major component of the fruit extracted by optimized to get clearer peaks. The resulting was ethanol which might lead to better 11 different peaks, the first peak was identified understanding for the pharmaceutical to be the peak of ethanol because of the importance of the fruit. concentration which is around 99.8%. For the HPLC analysis, the chromatogram (fig. 3) The peaks were predicted to be corresponding shows 17 peaks, peak number 5 identified to be 10 different compounds; mostly alkaloids the solvent peak because of its major because the test was run using alkaloid concentration of about 72.8%. The other peaks chromatography column for better are the peaks of 16 different compounds. The identification. The relative composition of the relative composition of the compounds was extracted alkaloids was calculated and the major calculated neglecting solvent peak, the first 4 peak which is corresponding to the main compounds accounted for major concentration component of the ethanol extract was the peak of about 84.3% of the whole concentration. The number 4 (retention time 20.6) with greatest peak was the peak number 4 (retention concentration of about 64.4% followed by peak time 6.00) with concentration 28.0% followed by number 9, 5, 2, and 3 respectively with peak number 1 (retention time 5.37) with concentrations ranging between 6%-8% while concentration of 27.5% followed by number 2 the peaks with number 6, 7, 8, and 10 had (22.8%) and number 3 (6.0%). The other 12 concentrations lower than 2.5% which accounts peaks of retention time ranging between 7-11.5 for other minor components. account for minor components with The identification of the peaks was not concentration < 2.5% including four trace performed because of the lack of standards for components with concentration < 0.3%. measuring the retention index of the alkaloid PAHs were identified and their concentration peaks, further research is needed for was also obtained by further analysis of the HPLC identification of these compounds. A standard of test results showing the presence of 6 PAHs from n-alkanes might be added to the extract to total of 16 most important PAHs with total

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concentration of about 411.6 nanogram per well as some trace compounds, as analyzed gram of extract as shown in table 5. with HPLC test. The following 6 PAHs were identified: Flourene, 3. Six toxic polycyclic aromatic hydrocarbons Phenanthrene, Flouranthene, Pyrene, are present with total concentration of about Benzo[a]anthracene, and Chrysene with 411.6 ng/g as found from the HPLC results. concentrations shown in the table 5. Table 6 4. Two confirmed human carcinogenic shows the structural formulae of these PAHs compounds are among the PAHs; ordered according to their concentration as well Phenanthrene and Flouranthene. as their carcinogenic and other health effects, 5. Two probable carcinogenic factors; Pyrene the classifications mentioned are all as described (highest concentration) and Chrysene are by the American Conference of Governmental also among the identified PAHs. Industrial Hygienists TLVs and BEIs. It shows that the ethanol extract contains two ACKNOWLEDGMENTS confirmed carcinogens; Phenanthrene and The authors are thankful to the headmistress of Flouranthene and two probable carcinogenic the gifted students’ school of Basra; Mrs. Ibtehal factors; Pyrene (highest concentration) and A. Alasady for providing facilities and support to Chrysene and two other PAHs that have no carry out the work. The authors are also thankful confirmed carcinogenic effect. to Lect. Ass. Salah M. Saleh and Lect. Ass. Abdulhusain A. Khwadem in the Marine Sciences CONCLUSION Center for providing the GC and HPLC tests and Plants are used widely in traditional medicine to Dunya K. Ahmed in the chemistry laboratory and they are sources for many powerful and of the gifted students’ school/ Basra for potent drugs. At the same time, they might providing lab assistance. contain some cancerous or toxic materials. Starting from these two points, phytochemical CONFLICT OF INTEREST STATEMENT studies of plant extracts have recently been a The authors declare that they have no very important and vital area of research in competing interests. order to determine these effects. In this study, the ethanol extract of Cordia myxa REFERENCES L. dried fruit was prepared and was then treated 1. Sathyaprabha G, Kumaravel S, Ruffina D, for dehydration, degasification and purification. Praveenkumar P. A comparative study on The ethanol extract was analyzed by using two antioxidant, proximate analysis, different techniques: gas chromatography for antimicrobial activity and phytochemical alkaloids and high performance liquid analysis of Aloe vera and Cissus chromatography for PAHs test. Some concluded quadrangularis by GC-MS. J Pharma 2010; remarks are mentioned below: 3(3):2970. 1. At least ten different alkaloids are present in 2. Harborne JB. Phytochemical methods: A the fruit extract with one alkaloid accounting guide to modern techniques of plant for about 64% of the fruit extract as found by analysis. New York: Springer; 2013. GC test; can be predicted to have lower 3. Schmelzer GH, Gurib-Fakim A. (Eds.). Plant molecular masses. resources of tropical Africa 11(1). Medicinal 2. At least sixteen compounds are present in the plants 1. PROTA foundation. Wageningen, extract with four major compounds Netherlands: Wageningen, Netherlands/ accounting for about 84% of concentration as

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Cite this article as: Tarik Z. T. Shwaish, Prof. Dr. Faris J. M. Al-Imarah. Chromatographic Study of the Ethanol Extract of Cordia Myxa L. Fruit. J Pharm Chem Biol Sci 2016; 4(2):142-152.

J Pharm Chem Biol Sci, June-August 2016; 4(2):142-152