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Medicinal chemistry
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Isolation and Structural Elucidation of 20 Hydroxyecdystone from Vitex doniana Sweet Stem bark (Black plum) Mustapha

Tijjani A1*, Shettima YA2, Abdulrahman FI1, Khan IZ1, Tom GM3 and Cong LI4

1Department of Chemistry, Faculty of Science, University of Maiduguri, PMB 1069, Maiduguri, Borno State, Nigeria

2Department of Biochemistry, Faculty of Science, University of Maiduguri, PMB 1069, Maiduguri, Borno State, Nigeria

3Departement of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Maiduguri, PMB 1069, Maiduguri, Borno State, Nigeria

4National Centre for Natural Products Research and Developement, Faculty of Pharmacy, University of Mississipi, United States of America

*Corresponding Author:
Tijjani A
Department of Chemistry
Faculty of Science, University of Maiduguri
PMB 1069, Maiduguri, Borno State, Nigeria
Tel: +2348000000000
E-mail: [email protected]

Received Date: March 22, 2017 Accepted Date: March 30, 2017 Published Date: April 03, 2017

Citation: Tijjani A, Shettima YA, Abdulrahman FI, Khan IZ, Tom GM, et al. (2017) Isolation and Structural Elucidation of 20 Hydroxyecdystone from Vitex doniana Sweet Stem bark (Black plum) Mustapha. Med Chem (Los Angeles) 7: 828-831. doi: 10.4172/2161-0444.1000436

Copyright: © 2017 Tijjani A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Vitex doniana Sweet, a plant commonly known black plum, in English, Prunier noir in French, dinya in Hausa, ucha koro in Igbo, oori-nla in yoruba and ngarmi in Kanuri is a medium-sized deciduous tree, 8-18 m high, with a heavy rounded crown and a clear bole up to 5 m. V. doniana is from Verbenaceae family and abundantly occurring in savannah regions. It can be found throughout tropical Africa. The ethanolic extract of Vitex doniana stem bark (11.9 g) was subjected to a silica gel accelerated column chromatography and eluents fractions (150 ml aliquots) obtained were collected and monitored with thin layer chromatography (TLC). Fractions with similar Rf values from same solvents system were poled together. Phytochemical test of all the fractions were perform. Complete elution yielded 48 fractions (150 ml/fraction) which were pooled to 24 fractions and finally to eight (8) eight fractions and coded. Fraction Vd8-a (56 mg) has gave a single spot a white crystal compound coded V1 on checking with TLC and observed under Ultraviolet lamp. The Rf values was calculated to be 0.433 and melting point was found to be 241-243°C uncorrected. The infrared spectrum of compound V1 shows prominent peaks that corresponds to OHstr (3365 cm-1) and C=0 (1652 cm-1). The 1H NMR (400 MHZ) spectrum of compound V1 in DMSO-d6 displayed five singlet signals. It further showed a broad singlet at δ 5.58 integrated for 1 H is due to an olefinic H-atom adjacent to the carbonyl carbon atom. Three signals at δ 3.10` (d, J = 9.0 Hz, H-22), 3.59 (m, 1H, 2H-a) and 3.72 (m, 1H, 3H-e) each integrating for one proton is due to an oxymethine protons indicating that three oxymethine H-atoms were present in the compound. The 13C-NMR spectrum showed the presence of 27 Carbon atoms, suggesting that may be steroid skeleton and the DEPT-135 spectra showed the presence of five CH3, eight CH2, and seven CH groups, and seven quaternary C-atoms. The Molecular formula was established as C27H44O7 by HRES-MS positive ion mode m/z 481.3179. Based on the spectral analysis, the compound V1 is thus concluded to have ecdysteriod skeleton and conclusively conforms with 2β, 3β 14α, 20R, 22R, 25- hexahydroxy-5 β cholest- 7-ene-6- one, commonly known as 20-hydroxyecdysone. This is the first time this compound was isolated from Vitex doniana sweet.

Keywords

Vitex; Phytochemical; Purification; Isolation; Chromatography; Spectroscopy

Introduction

Nearly all cultures of the world, both ancient and recent have heavily depended on plants as a therapeutic agent used in various forms. Plants play a major role in the treatment of diseases and still remain the foremost alternative for a large majority of people [1]. The knowledge of plants if wisely utilized could bring out promising herbal leads. The World Health Organization (WHO) has reported that about 80% of the world population relies on traditional medicine to cure ailments [2,3]. Plants are used directly as therapeutic agents, as well as starting material for the synthesis of drugs or as models for pharmacologically active compounds [4]. The study of natural products has had a number of rewards. It has led to the discovery of a variety of useful drugs for the treatment of diverse diseases and contributed to the development of spectroscopic methods of structure elucidation and synthetic methodologies that is now the basis of analytical organic chemistry. Vitex doniana is a medium-sized deciduous tree, 8-18m high, with a heavy rounded crown. The V. doniana bark is rough, pale brown or greyish-white, rather smooth with narrow vertical fissures. Vitex doniana Sweet commonly known as black plum, in English, Prunier noir in French, "dinya" in Hausa, "ucha koro "in Igbo, " oorinla" in yoruba and "ngarmi" in Kanuri. Vitex doniana can be found in deciduous forest, coastal woodland, riverine and lowland extending as high as upland grassland, secondary and dry forests. It can be found throughout tropical Africa [1,4].

Experimentation

Sample collection and identification

The stem bark of Vitex doniana leaves were collected in Kawuri village of Konduga Local Government Area of Borno state of Nigeria. The plant specimen was identified and authenticated by a Plant Taxonomist, Prof. S.S. Sanusi of the Department of Biological Sciences, Faculty of Science, University of Maiduguri. The herbarium specimen with a voucher number 555 C was deposited at the Post Graduate Research Laboratory, Department of Chemistry. The stem bark of the Vitex doniana was cleaned and air-dried in the laboratory. Two kilogram (2 kg) of the stem bark of Vitex doniana was pulverised into a coarse powder using mortar and pestle.

Extraction of stem bark of V. doniana and phytochemical analysis

The weighed powdered air-dried sample (2 kg) was macerated (cold extreaction) with 95% ethanol for five days filtered and evaporated in vacuo at 40°C using a rotary evaporator. The extract concentrate was labeled and the percentage yield were calculated in w/w. The ethanolic extract was subjected to qualitative chemical screening for identification of the primary and secondary metabolites such as flavonoids, alkaloids, sterols, triterpenes, saponins, anthracenosides, tannins, polyuronides, emodol, as described by Safowora and Evans [5-7].

Isolation of compound from ethanolic extract of V. doniana stem bark

The ethanol extract (11.9 g) was fractionated on a silica gel column chromatography using solvents such as n-hexane, ethyl acetate and methanol. Three hundred grams (300 g) silica gel 60-120 mesh was packed manually into a column (75 cm × 3 cm). The silica gel was first swell in n-hexane and filled into the column to a bedding height of 60 cm and then allowed to settle and packed. The ethanolic extract was then dissolved in 7 ml of n-hexane, mixed with silica gel subsequently applied on the top of the column. The ethanolic extract was gradiently and successively eluted with n-hexane, ethyl acetate and methanol 100% at a flow rate of 1ml/min. Each eluents fraction (250 ml aliquots) was collected and monitored with thin layer chromatography by making slurry of silica gel 60G merck, Germany on 20 cm × 20 cm and 7 cm × 5 cm precoated silica gel TLC plates. Fractions with similar Rf values from same solvents system were poled together. Phytochemical test of all the fractions were performed using standard procedure. The melting points of the isolated compounds were determined using Gallenkamp capillary melting point apparatus and the results were uncorrected.

Characterization of the isolated compound coded V1

Compound V1 was subjected to structural elucidation using infra-red spectroscopy, Nuclear Magnetic resonance (both 1H and 13C) and Mass spectroscopy. The Infra-red (IR) spectra of the isolated compound was recorded in a thin film in Nujol using Genesis series Fourier transform-infrared spectrophotometer (FT-IR), HNMR (one and two dimension spectra) was recorded using 400 MHz (Varian Instruments) DRX-500 spectrometer. 13C NMR was recorded using variant instruments 100 MHz. High- resolution TOF- MS were measured on an Aglient series with and ESI (HRESI-MS).

Results and Discussion

Isolation of compound from V. doniana stem bark

Complete elution yielded 48 fractions (150 ml/fraction) which were pooled to 24 fractions base on the Rf values. It was further recombined and 12 fractions were obtained on the basis on Rf values and coded Vd1 to Vd12 fractions. Vd8 was further eluted with ethylacetate and methanol and gave fourteen 14 sub fractions Vd8-a, to Vd8-m. Fraction Vd8-a (56 mg) has gave a white crystal compound coded V1. It was further checked on TLC solvent system (Hexane, ethylacetate and methanol 7:3:1) and was found to give a single spot. The Rf values was calculated to be 0.433. The melting point was determined to be 241-243°C uncorrected.

Phytochemical analysis of fractions fractions Vd1 to Vd12

The result of the preliminary phytochemical analysis is shown here below in Tables 1-4. The result indicates that only fraction Vd1 shows positive test of flavonoid. Carbohydrates is present in Vd2 Vd5, and Vd10. Tannins is found in fractions Vd5 and Vd9 while steroidal nucleus is present in Vd2, Vd6 and Vd8. Terpenes is found in fractions Vd2 and Vd5 while soluble starch and saponins can be found in fractions Vd5 and Vd8 as well as fractions Vd1 and Vd12. Alkaloids and anthraquinones were absent in all the fractions.

Class of Chemical Components Vd1 Vd2 Vd3 Vd4 Vd5 Vd6 Vd17 Vd18 Vd19 Vd10 Vd11
Test of Alkaloids - - - - - - - - - - -
Test for Flavonoid + - - - - - - - - - -
Test for carbohydrates - - + - - + - - - + +
Test for tannins - - - - + - - - + - -
Test for free Anthraquinones - - - - - - - - - - -
Test for Steroidal nucleus - + - - - - - + - - -
Test for Terpenoids - + - - + - - - - - -
Test for soluble starch - - - - + - - - + - -
Test for saponins - - - - - - - - - - +

Table 1: Phytochemical analysis of ethanolic extract fractions Vd1 to Vd12.

Functional group Wave number cm-1
CH3Vasy 2962
Ohstr 3363
C=Cbend,str 1422
C=O 1652
C-Hbend 879.57

Table 2: Analysis of IR spectra of compound V1.

Chemical Shifts multiplicities (ppm)
Compound V1 in DMSO-d6 20 Hydroxy ecdysterod
0.72 (3H, S H-18) 0.75 (3H, S H-18)
0.79 (3H, S, H-19) 0.79 (3H, S H-19)
1.01 (3H, S, H-21) 1.03 (3H, S H-21)
1.03 (3H, S, H-26) 1.04 (3H, S H-26)
1.18 (3H, S, H-27) 1.06 (3H, S H-27)
2.99 (1H, m, H-19) 2.99 (1H, m H-19)
3.1 (d, j=10.0 Hz, H-22) 3.1 (d, j=10.0 Hz H-22)
3.50 (m, 1H, 2H-a) 3.5 (m, 1H, 2H-a)
3.75 (m, 1H, 3H-e) 3.75 (m, 1H 3H-e)
5.6 (m, 1H, H-7) 5.6 (IH, m H-7)

Table 3: H1 NMR Data for compound V1 in DMSO-d6 and compared with the Literature.

Carbon     Chemical Shift Multiplicities (d)
Compound V1 20 hydroxy ecdysteroid
C-1 36.6t 36.8t
C-2 66.8d 66.9d
C-3 66.7d 66.7d
C-4 31.7t 31.7t
C-5 50.5d 50.4d
C-6 202.8 202.9
C-7 120.5d 120.7d
C-8 165.3s 165.4s
C-9 33.2d 33.4d
C-10 37.7s 37.8s
C-11 20.3s 20.5s
C-12 31.5t 31.1t
C-13 46.9s 47.0s
C-14 83.0s 83.1s
C-15 30.0t 30.0t
C-16 20.3t 20.5t
C-17 50.1t 50.3t
C-18 17.2q 17.3q
C-19 23.9q 24.0q
C-20 75.7s 75.8s
C-21 21.0q 21.1q
C-22 69.0d 70.3d
C-23 26.1t 26.2t
C-24 41.5t 41.5t
C-25 68.8s 68.9s
C-26 29.1q 29.2q

Table 4: 13C NMR Data of compound V1.

Discussion on the characterization of compound V1

The Infra–red (IR) spectrum of compound V1 shows prominent peaks at 3363 cm-1, 2963 cm-1, 1652 cm-1 and 1421 cm-1. The peaks corresponds to OHstr (3365 cm-1), CH3Vas, (2962 cm-1), C=0 (1652 cm-1) and C=C bend,stre (1421). This spectrum suggests that among the functional moiety in compound V1 are the carbonyl and hydroxyl group. The UV spectra of compounds were consistent with the presence of an α, β- unsaturated ketone chromophore group.

The 1H NMR (400 MHZ) spectrum of compound V1 in DMSO-d6 displayed five singlet signals at δ 0.72 (3H, s, H-18), 0.79 (3H, s, H-19), 1.03 (3H, s, H-21), 1.04 (3H, s, H-26), 1.06 (3H, s, H-27) each integrating for three protons indicating the five methyl functional groups are present in the compound. It further showed a multiplet at δ 5.58 integrated for 1 H is due to an olefinic H-atom adjacent to the carbonyl carbon atom. Three signals at δ 3.10` (d, J=9.0 Hz, H-22), 3.59 (m, 1H, 2H-a) and 3.72 (m, 1H, 3H-e) each integrating for one proton is due to an oxymethine protons indicating that three oxymethine H-atoms are present in the compound. These all signals are characteristic to the ecdysteroid skeletons.

The 13C-NMR spectrum showed the presence of 27 Carbon atoms, suggesting that may be steroid skeleton. A signal at δ 202.77 (C-6) indicating that a carbonyl carbon atom is present in compound. Two signals at 165.32 (C-8) and 120.50 (C-7) due to an olefinic carbon atoms suggesting that double bond is present. Three signals at δ 83.03 (C-14), 75.74 (C-20), and 68.76 (C-25), is due to three quaternary oxygenated carbon atoms. Three signals at δ 76.23 (C-22), 66.82 (C-3), 66.63 (C-2) is suggesting the three oxymethine carbon atoms. Further, the 13C-NMR spectrum showed signals characteristic of ecdysteroid skeleton.

The DEPT-135 experiment showed the presence of five CH3, eight CH2, and seven CH groups, and seven quaternary C-atoms.

The DEPT spectrum peaks for the five CH3 at C-19 (24.1 ppm), C-21 (21.1 ppm), C-26 (29.2 ppm), C-27 (29.1 ppm) and C-18 (17.3 ppm). CH peaks are at C-2 (66.9 ppm), C-3 (66.7 ppm), C-5 (50.3 ppm), C-7 (120.7 ppm), C-9 (33.4 ppm), C-17 (50.3 ppm) and C-22 (76.3 ppm). The Spectrum further indicates that there are eight peaks at 36.8 ppm (C-1), 31.7 ppm (C-4), 20.6 ppm (C-19), 31.1 ppm (C- 12), 30.5 ppm (C-15), 20.5 ppm (C-16), 26.5 ppm (C-23), and finally 41.5 ppm (C-24). The quaternary carbon atom peaks at 202 ppm (C-6) which is the carbonyl atom, 165.4 ppm (C-8), 37.8 ppm (C-10), 47.0 ppm (C-13), 83.1 ppm (C-H), 75.8 ppm (C-20) and 68.9 ppm (C-25).

The 2-D NMR clearly indicated the connectivities between the carbon atoms in the skeleton of the molecule and between the C-H bonds as well as the position of the unsaturation. The heteronuclear multiple bond quantum coherence (HMQC) spectra indicated that there is an interaction between the proton at 0.72 ppm (H-18) and that of C-18 at 17.3 ppm. There is connectivity for proton H-19 at 0.79 ppm and C-19 at 24.19 ppm. Connectivties at 1.06 ppm for H-27 and 29.2 ppm for C-27 ppm was also recorded. Proton H-22 at 3.16 ppm is connected to C-22 at 76.3 ppm while proton H-2 and H-3 at 3.50 ppm and 3.75 ppm is connected C-2 and C-3 at 66.9 ppm and 66.9 ppm.

The Molecular formula was established as C27H44O7 by High resolution Electron spray ionization-Mass spectroscopy (HRESI-MS) positive ion mode m/z 481.3179 [M+H]+ . The signals in Mass spectrum 463, 445, and 427 peaks corresponding to losses of one, two, three, or four water molecules are characteristic for Ecdysterone skeleton reported in the literature. In the commonly used electron- impact (EI) mode, a mass spectrometer bombards molecules in the vapour phase with a high-energy electron beam and records the result of the electron impact as a spectrum of positive ions separated on the basis of mass/ charge (m/z); most of the ions are singly charged.

Based on the spectral analysis (1H NMR, 13C NMR, DEPT, HMQC, IR, HRESI-MS) the compound V1 is thus concluded to have ecdysteriod skeleton and conclusively conforms with 2β 3β 14α, 20R, 22R, 25- hexahydroxy-5β cholest-7-ene-6- one, or 2, 3,14, 20, 25 Hexa hydroxyl cholest-7-ene-6-one commonly known as 20-hydroxyecdysone as shown in Figure 1. The structure was earlier established by Zhu et al. and Bathori et al. in other plants [8,9]. However, this is the first time this structure or compound was established in Vitex doniana sweet. Over 250 ecdysteroid analogs have been identified so far in plants, and Dinan et al. [10] has indicated that there are over 1,000 possible structures which might occur in nature. 20-hydroxyecdysone, possessing the cholesterol nucleus, is present in Ajuga turkestanica, Vitex glabrata), Tapinella panuoides [10].

medicinal-chemistry-hydroxyecdystone

Figure 1: 2ß 3ß 14a, 20R, 22R, 25- hexahydroxy-cholest-7-ene-6- one (20 hydroxyecdystone).

Acknowledgements

We wish to acknowledge the technical support of Mr. Fine Akawu of Department of Chemistry, University of Maiduguri and Dr. Ranga Roa Ravu of National Centre for Natural Products Research and Developement Faculty of Pharmacy, University of Mississipi, United States of America.

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