Phytochemical Investigation and Characterization on the Root Bark Extract of Prunus Africana

Prunus africana is one of the most popular to treat benign prostate hyperplasia (BHP), and to treat diarrhoea, dysmenorrhoea, infertility, irregular menstruation, kidney disease, disorders, fevers, obesity, pneumonia, hypertension, antigonorrheic, antimalarial, chest pain; other various diseases. The air dried and powdered plant material (200g) was first soaked with 500 mL n-hexane for 72 hours and yielded 1.3 g of n-hexane extract. Marc was soaked with 400 mL of ethyl acetate for 72 hours and afforded 2g of ethyl acetate extract. Finally, Marc was soaked with 400mL of methanol and yielded 12.6 g of methanol extract. The ethyl acetate extract of the root bark of P. africana afforded a compound coded as AYU . Its Structural determination was accomplished by means of spectroscopic techniques, namely IR, 1 H NMR, 13 C NMR and DEPT-135. The compound, AYU was isolated and characterized from the root bark of P. african . It was shown that spot on TLC only up on spraying 1% vanillin sulphuric acid and after heating for a few minutes. Generally, chromatographic techniques like PTLC and HPLC are required to isolate more compounds from leaves of P. africana . Also MS and 2D NMR spectroscopic techniques are needed to fully characterize the isolated compound.


Uses of P. africana with Exploitation
The discovery of the medicinal properties of Prunus bark initiated a decades' long harvest for international market needs, in which Cameroon supplied over half, followed by Madagascar, the Democratic Republic of the Congo (DRC), Kenya, Uganda, and Equatorial Guinea. 7 For the last 35 years, the African cherry (P. africana) has been used in the treatment of benign prostatic hyperplasia and other disorders. The bark, from which the treatment is derived, is entirely wild-collected. The major exporters of bark include Cameroon, Madagascar, Equatorial Guinea, and Kenya. To the group of Fournier France, Indian, and Italy which exported 86% of the world bark extracts. Worldwide exports of dried bark in 2000 have been estimated at 1350-1525 metric tons per year, down from its peak of 3225 tons in 1997. Bark extracts (6370-7225 kg per year) are worth an estimated $ 4.36 million per year. 11 While, in Ethiopia, the African cherry is basically used for fuel wood, charcoal, ornamental, windbreaks and green manure. 12 Bark infusions were used in the treatment of chest pains and as a purgative for cattle. 13 In Africa, 500 tonnes of bark were harvested for use in traditional medicine annually. 14 The use of the species in pharmaceutical industry commercially began in 1970s. In 1980, only 200 tonnes of P. africana bark were harvested. 15 The demand for the bark in pharmaceutical industry increased from then and by 1997 the demand had risen to 3500 tonnes. 16 This involved processing and marketing of bark and bark extracts for the treatment of benign prostate hyperplasia. 17 Benign prostatic hyperplasia (BPH) is a condition common in most men and manifests itself as increased frequency in urination, pain in passing urine, inability to empty the bladder and post urinary dribbling. 18 Phytosterols eliminate vassal congestion and excess blood hence reduces the size of prostate adenomas. The pentacyclic triterpenoids block enzymatic activity consequently inhibits inflammation in the prostate. 19,20 Thus, the study was aimed at phytochemical investigation and characterization, on the root bark of P.africana.

Plant Material
The root bark of P. africana was collected from Shero kebele borderline of Abose and Weshiso land, Misha Woreda, Hadiya Administrative Zone, Southern Nations Nationalities and People Regional State. 1 H and 13 C NMR spectra were recorded on a Bruker Advance 400 MHz spectrometer in CDCl3 with TMS as internal started. The ultra-violet and visible (UV-Vis) spectra were taken on GENESY'S 2PC UV-Vis scanning spectrometer (200-800 nm). IR spectra were obtained on Perkin-Elmer BX infrared spectrometer (400-4000 cm -1 ) using KBr. Melting point was recorded using digital melting point apparatus. Analytical thin layer chromatograms were run on 0.2 mm thick layer of silica gel coated on aluminum foil. Column chromatography was performed using silica gel (70-230 mesh). List of solvents were used in this research like: n-hexane, ethyl acetate, methanol and others. Some of the apparatus were used: funnels, round bottom flasks, vials, glass wares, refrigerator, Whatman No.1 filter papers, grinder, drying oven, measuring cylinders, TLC Chamber, capillary tubes, cuvette quartz etc...

Extraction and Isolation
The air dried and powdered plant material (200g) was first soaked with 500 ml n-hexane for 72 hours and the Chemistry and Materials Research www.iiste.org ISSN 2224-3224 (Print) ISSN 2225-0956 (Online) Vol.12 No. 6,2020 extract was collected by filtering and concentrated under reduced pressure using the Rotavapor and yielded 1.3g. The solvent free Marc was then soaked with 400 ml of ethyl acetate for 72 hours and the extract was collected. This filtrate was evaporated under reduced pressure using the Rotavapor and afforded 2g. Finally, the solvent free Marc was soaked with 400 ml of methanol, and then it was filtrated by using Whatman no.1 filter paper and concentrated under reduced pressure using the Rotavapor and it was yielded sample of 12.6g, and its extract was afforded many spots on TLC. Solvent was removed using rotary evaporator. Compound on TLC was detected after spraying 1% vanillin sulphuric acid and after heating for a few minutes. There was no visible spot for n-hexane extract, but ethyl acetate extracts were showed three colored spots by using solvent system of n-hexane: ethyl acetate (6:4), and its yield of 2g crude extract was dissolved in itself with equivalent amount of silica gel, dried using Rotavapor and applied to a silica gel (200g) column chromatography which was packed with n-hexane (100%). Table1 The numbers of fractions were collected. Out of these fractions which were collected using the solvent systems increased polarity, some of them were showed the characteristic colored spots on TLC up on spraying 1% vanillin sulphuric acid and after heating for a few minutes. The remaining fractions did not show the characteristic colored spots on TLC up on spraying 1% vanillin sulphuric acid and after heating for a few minutes. Among fractions, fraction 58 showed single spot on TLC using the solvent system n-hexane: ethyl acetate (6:4) upon spraying 1% vanillin sulphuric acid and after heating for a few minutes. Finally, the dried sample of this fraction was afforded 17mg of the compound, AYU.

Phytochemical Screening of root bark extract of P. africana
The extracts phytochemical analysis for identification of chemical constituents was done using standard procedures from literature. 1. Tannins: About 0.1g of the extract was put in a test tube and 20mL of distilled water was added and heated to boiling. The mixture was then filtered and 0.1 % of FeCl3 was added to the filtrate and observations made. A brownish green color or a blue-black coloration indicated the presence of tannins. 2. Saponins: About 0.1g of the extract was mixed with 5mL of water and vigorously shaken. The formation of stable form indicated the presence of saponins. 3. Flavonoids: About 0.1g of the extract was added in to a test tube. To the test tube 5mL of dilute ammonia and 2mL of concentrated sulphuric acid was added and heated for about 2 minutes. The appearance of a yellow color indicated the presence of flavonoids. 4. Terpenoids: About 0.1g of the extract was taken in a clean test tube; 2mL of chloroform was added and vigorously shaken, then evaporated to dryness. To this, 2mL of concentrated sulphuric acid was added and heated for about 2 minutes. A grayish color indicated the presence of terpenoids. 5. Glycosides: About 0.1g of the extract was mixed with 2mL of chloroform and 2mL of concentrated sulphuric acid was carefully added and shaken gently, then the observations were made. A red brown color indicate the presence of steroidal ring (glycone portion of glycoside) 6. Alkaloids: About 0.1g of the extract was mixed with 1% of HCl in a test tube. The test tube was then heated gently and filtered. To the filtrate a few drops of Wagner's reagents were added by the side of the test tube. A resulting precipitate confirmed the presence of alkaloids. 7. Steroids: About 0.1g of the extract was put in a test tube and 10mL of chloroform added and filtered. Then 2mL of the filtrate was mixed with 2mL of a mixture of acetic acid and concentrated sulphuric acid. Bluish green ring indicated the presence of steroids. 8. Phenols: About 0.1g of the extract was put in a test tube and treated with a few drops of 2% of FeCl3; blue green or black coloration indicated the presence of phenols. 21 The air dried and powdered leaves of the P. africana (200g) were extracted with solvents of n-hexane, ethyl acetate and methanol. These extracts when developed on TLC both the n-hexane and ethyl acetate extracts have shown three colored spots, but methanol extract was afforded many spots on TLC. The orange organic extract of ethyl acetate (2g) was subjected to column chromatography on silica gel.

Characterization of Compound, AYU
The compound, AYU was obtained as a white solid that showed a characteristic color change to violet on TLC plate upon spraying 1% vanillin sulphuric acid and after heating for a few minutes. It has retention factor, RF value 0.8 using hexane: ethyl acetate (6:4) as solvent system. In the IR spectrum of the compound, AYU the absorption band at 3400cm -1 showed the O-H stretching that indicated the presence of a hydroxyl group. The strong absorption band at 2922cm -1 showed the presence of the C-H stretching of the olefinic group. The weak absorption band at 2850cm -1 showed the presence of the C-H stretching for sp 3 groups. Two strong absorption band at1690cm -1 and 1609cm -1 showed the presence of the olefinic C=C stretching. The absorption band at 1170cm -1 showed the presence of the C-O bond stretching. proton groups and assigned to C-1. Coupled proton peaks, which is triplet peaks at δ 2.10; 1.99, integrating for two protons, which were corresponded to the methylene proton groups and assigned to C-2. The pentet peaks at δ 3.41, integrating for one proton, corresponded to the methine proton group that assigned at C-3. Coupled proton peaks, which is triplet peaks at δ 1.89; 1.60, which integrating for two protons corresponded to the methylene proton groups and attached to C-4. Triplet peaks at δ 1.90, integrating for one proton corresponded to the methine proton, which were assigned to C-5. The quartet peaks at δ 3.3, integrating for one proton, corresponded to the methine proton and assigned to C-9. Coupled proton peaks, which is doublet peaks at δ 2.30; 2.10, integrating for two protons corresponded to the methylene proton which was attached to C-11. The pentet peaks at δ 2.10 which integrating for one proton corresponded to the methine proton and attached to C-12. Coupled proton peaks, which is doublet peaks at δ 2.30; 2.10, which integrating for two protons corresponded to the methylene proton and attached to C-13. Singlet peak at δ 1.20 which integrating for six protons corresponded to the methyl protons and attached to C-14 and C-15. Doublet peaks at δ 1.10, which integrating for three protons corresponded to the methyl protons and attached to C-16. Doublet peaks at δ 1.23, which integrating for three protons corresponded to the methyl protons and it attached to C-17.
The 13 C NMR and DEPT-135 spectrum of compound, AYU showed well resolved resonance of 17C atoms of which 4, 4, 5, and 4 of them were methyl, methylene, methine, and quaternary carbon groups, respectively.  13 CNMR and DEPT-135 spectrum of compound (AYU) showed well resolved resonance of 17 carbon atoms and from them were: four methyl groups, four methylene groups, five methine groups and four quaternary carbons.  Fig. 1. The proposed structure of the compound, AYU Key: AYU= code, which was given for proposed structure, there is no other meaning

Conclusions and Recommendations
P. africana is a commercial by its stem bark which is most popular to treat benign prostate hyperplasia (BHP). In the this study, root bark of p.africana were showed the presence of phytochemical constituents such as alkaloids, flavonoids, terpenoids, saponins, tannins, steroids, and phenols of ethyl acetate extracts of it and also confirms that the absence of saponins, terpenoids, and steroids in the hexane extracts of P.africana root bark. The root bark of P.africana was extracted with solvents of n-hexane, ethyl acetate, and methanol and their yields 1.3g, 2g, and 12.6g, respectively. The orange organic extract of ethyl acetate (2g) was subjected to column chromatography on silica gel and numbers of fractions were collected. That is, from the IR spectrum, the absorption band at 3400cm -1 shows the O-H stretching that confirms the presence of a hydroxyl group. Also the strong absorption band at 2922cm -1 shows the presence of the C-H stretching. Two strong absorption band at 1690cm -1 and 1609 cm -1 shows the presence of the C=C stretching of the olefinic group.
As a final point, from this study, the compound AYU was elucidated and characterised by incorporating by spectroscopic techniques such as IR spectral data, 13 C NMR, DEPT-135, and 1 H NMR spectral data obtained.
Despite the traditional use of this plant for the treatments of various ailments, in many parts of the world there is no more report on phytochemical analysis on the root bark of P. africana. This is provoked tricky to compare and contrasts my work with the relative of other work. Thus, this study may serve as baseline for researchers who are inspired and interested to conduct such type of research in the future.