Oguegbulu N Edwin and Uche I Fidelia.

/ JPBMS, 2012, 15 (14)

Available online at www.jpbms.info

Research article JPBMS

ISSN NO- 2230 7885 CODEN JPBSCT

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Extraction and detection of the quarternary alkaloids of the stem bark of Alstonia boonei plant (family: apocynaceae), by the sulphonephthalein dye-stuff ion-pair complexation at pH 5.58.
*Oguegbulu, N. Edwin1, Uche, I.Fidelia2
1Department

of Pharmaceutical and Medicinal Chemistry,Faculty of Pharmaceutical Sciences, University of Port Harcourt, Rivers, State Nigeria. 2Department of Pharmacognosy and Phytomedicine, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Rivers, State Nigeria.

Abstract:
Background and aim: Alkaloids as vegetable alkalis found often in higher plants, are however occasionally present also in lower plants and in animals. They are indeed very medicinally useful secondary metabolites and can occur as primary, secondary, tertiary or quaternary compounds. The extractability of quaternary alkaloids as in N-oxides tends to pose a serious challenge to natural products Chemists, especially in the resource - limited countries. This is because of their high level of polarity and hence a significant affinity to the aqueous-acid phase usually employed in the common extraction process. Alstonia boonei, a proven versatile medicinal plant used in African traditional medicine has been confirmed to contain a heavy load of alkaloids characteristics of which are consistent with the already established quaternary alkaloids of the other members of the Alstonia species. The objective of this research is to address these highlighted challenges posed by the extractability complexity of quaternary alkaloid and to aid in the quick identification of the same in the far away wood. Methodology: Complexation of these quaternary alkaloids with bromocresol purple a sulphonephthalein dye, at a specific pH of 5.58 was achieved and subjected to a comparison with Zn-reduction extracts of same using tlc techniques. Results: The observations have led to a prospect for enhanced and quick extractability of quaternary alkaloids of this plant using simple facilities of; TLC, UV lamp and chromatographic colour reactions for the detection. Conclusion: The dye stuff-complexation extracts have compared favourably with the extraction product of Zn-reduction technique already established for the quaternary alkaloidal extraction. Development of this method can then be a fast, simple and reliable innovation in the extraction of quarternary alkaloids of Alstonia boonei

Key Words: Alstonia boonei plant stem bark, quaternary alkaloids, Zn reduction complexes,bromocresol ion-pair
complexation extracts at pH 5.58, tlc detection.

Introduction:
The term alkaloids can apply to the naturally occurring organic base containing one or more heterocyclic nitrogen atoms in the molecule. This concept was first proposed by a Pharmacist, W. Meissner in 1819 to cover a group of naturally occurring substances, which were vegetable alkalis. Alkaloids are primarily found in higher plants but also in lower organisms and even in some animals. Alkaloids too exhibit significant pharmacological activity. They contain some of the most complicated molecular structures and represent one of the largest and most diverse families of natural compounds. Alkaloids also represent the oldest drugs, and since the time of Hypocrites, their extracts from plants have been known to serve as medicines for a number of diseases. Alkaloids are known to be derivable from certain amino acids, pyridine nucleotide pool, monoterpenes, sesquiterpines, triterpenes and sterols. A smaller number is derived from athranilic acid and pyrimidine bases. Part of the nicotinic acid family
1

of alkaloids, all of the isoprenoids alkaloids and the purine derivatives are sometimes referred to as pseudo alkaloids or alkaloida imperfecta. In several examples, an alkaloid can accumulate in concentrations up to 10% of the dry weight of the substance [1]. Natural products are good objects of investigation because, according to WHO ,up to 80% of the worlds population depends on traditional medicine for its primary health care needs and the world market for herbal medicines including herbal products and raw materials has been estimated to reach US $43 billion with an annual growth rate of between 5 and 15%[2]. Alkaloids are often extracted by an acid-base shake out technique. However, according to Phillipson, Quaternary alkaloids cannot be extracted easily by these methods because; in general they remain in the aqueous alkaline phase when partitioned with immiscible organic solvent. If

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the aqueous alkaline phase is acidified with mineral acid, then quaternary alkaloids can be precipitated [3]. Alstonia boonei (family: Apocynaceae) is a tropical plant with versatile therapeutic applications in the traditional medicine [4], and with some scientific validations [5- 7]. It is thus used as; bitters and astringents, fever remedy, dysmenorrhoea, fibroid, anticonvulsants, pains, tooth ache, relaxation of spasms, malaria, chronic diarrhea, anti- snake venom, expulsion of placenta, anti-inflammatory, treatment of certain types of female infertility and antibacterial activity. The nitrogen atom of any alkaloid can occur as primary (mescaline), secondary (ephedrine), tertiary (atropine) or quaternary (the nitrogen atoms of tubercurarine and those found in Alstonia species as in figures; I, II and III [8].
N+

Experimental:
Dry and pulverized stem bark of Alstonia boonei plant (Family: Apocynaceae), collected by Mr.O.U Etefia of Pharmacognosy Department of University of Uyo, from Item junction community in Ikono Local Government area of Akwa Ibom State, Nigeria and identified b a Taxonomist, Dr. (Mrs.) Uduak A.Eshiet in the Department of Botany of University of Uyo, Akwa Ibom State of Nigeria. This was thereafter subjected to preliminary phytochemical tests following the standard method [10]. This confirmed the presence of alkaloids, glycosides (cardiac, cyanogenic and steroidal aglycones), saponins and tannins. This was further defatted with N-hexane prior to maceration with ethanol for three consecutive days with continuous agitation to ensure proper percolation. The alcoholic extract was concentrated by air - drying at ambient laboratory temperatures and then the alkaloidal constituents taken up in dilute acid solution (5% HCl). At this point, the neutral organic materials such as; some pigments and lipids were eliminated. The quaternary alkaloids present were reduced by addition of Zinc dust following a reported technique [11], just as they formed Iron-pair complexes or associates with a sulphonephthalein dye stuff [12,13]. Bromocresol purple (B.P) was used in this study. The aqueous acid fraction which notably was unextractable in CHCl3 prior to this complexation treatment was labeled as follows; A Bromocresol purple in aqueous acid fraction (+ ve for alkaloids) at pH 5.58 and then partitioned with CHCl3 in a separatory funnel, produced an orange colour and designated as sample A. B - Bromocresol purple in aqueous acid fraction as in A above at pH 7.23 and partitioned with CHCl3 as in A too; produced a yellow colour and labeled as sample- B C - Zn reduction product, which confirmed the presence of N-oxide (Quaternary alkaloids) and labeled as sampleC.

N
O
Alstonine (A. Constricta)

N+
H

Om

N
O
Alstoniline (A. Constricta)

H OO CooMe
H CooMe

Co oM

II

N 1
H

0 1
H
Alschonine (A. Scholaris)

0
+ NO

III

Figures I, II and III: Structures of some quaternary alkaloidal constituents of Alstonia Species

Several Alstonia species (family: Apocynaceae) have been useful medicinally. They include; Indian Alstonia scholaris and the Australian Alstonia constricta. Several of them are known to contain in common, a number of quaternary alkaloids some of which are shown in figures; I, II and III above. According to Trease et al., Although the formation of Noxides and N-oxidation products of alkaloids in animal systems is well known, forming part of the wider scheme for the metabolism of animals, the occurrence of such compounds in plants has until recently, received little attention. This is possibly due to a belief that such compounds represented artifacts arising during the extraction and work up of tertiary alkaloids. Secondly, because of high polarity and water solubility of alkaloid Noxides, they were discarded by the normal alkaloid extraction procedure [9]. The nature of the nitrogen atoms of the alkaloid can then determine the type of corresponding derivatives to be prepared and hence the isolation procedures to be adopted.
2

Detection of extractable components.

The trial tlc in various solvent systems indicated that Nbutanol: acetic acid: water (BAW) at (4: 1:5) gave the best resolution for the CHCl3 extracts. The samples labeled; A, B and C were further subjected to tlc using standard procedure [14]. The profile of the chromatogram is as represented in figure. II below.
Solvent front Orange Colour of dye X X

Figure II; The chromatogram of samples A, B and C in BAW (4:1:5)

3 - Dye + ext CHCl (PH 7.23) Sample B

CHCl3- Dye + ext (PH 5.58) Sample A CHCl - Zn + ext 3 (Sample C)

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The chromatogram was viewed under a U.V lamp (PW Allen and co UK.) for a delineation of the spots. Reactions with iodine vapour which stained orange and that with Dragendorff spray reagent which tested positive for alkaloids by staining brownish colour were indicative of successful extraction of alkaloids in samples A & C but not B. Samples A and C again compared favourably with respect to chromatographic reagents colour changes as well as in their Rf values and this varied significantly with sample B where the chromatographic profile was inconsistent with product of Iron pair complexation at pH 5.58, thereby depicting no alkaloidal extraction.

potential of enhanced extractability of the quaternary alkaloid of this important plant- Alstonia boonei.

Acknowledgement:
The authors owe their debt of gratitude to all the staff members of Departments of Pharmaceutical and Medicinal Chemistry as well as Pharmacognosy and Natural Medicine of Faculty of Pharmacy, University of Uyo, Akwa Ibom State, Nigeria.

References:
1.Waller GA, Nowack EK.Alkaloid Biology and Metabolism in Plants. Planum Publications. N.Y. 1978. 2.WHO. Report of the Inter Regional Workshop on Intellectual Property Rights, in the context of Traditional Medicine, WHO/EDM/TRM/2001.1.WHO Geneva.2001 3.Phillipson JD.Alkaloids.ThePharm.Journal.Jan, 29.1983. 102-3. 4.ParzialeE.EarthnotesHerbLibrary.http://earthnotes.tripo d.com/alstonia.htm.2001. 5.Omoregbe RE, Ikuebe CM, Ihimire 1G.Antimicrobial activity of some medicinal plants extracts on Escherichia coli, Salmonella paratyphi and Shigella dysenteriac. Afr. J. Med. & Med Sci.1996.25 (4):373-5. 6.Akah PA, NwambieAI.NigerianPlantswithanticonvulsant property. Journal Fitoterapia.1993.64:15-120. 7.Osadebe PO.Anti-inflammatory properties of the root bark of Alstmia boonei, Nig. Formal of Nat. Prods. And Med. 2002. 6: 39-41. 8.Buckingham J (Ed) .Dictionary of Natural Products. Vol. 1- A.C. 5th edn .Chapman and Hall 3rd Ave. N.Y. PP 187-8. 1994. 9.Trease EG, Evans WC.Pharmacognosy.11th edn. Bailliere Tindall. London.1978. 10.Harbone JB.: Phytochemical methods. 2nd edn. Chapman and Hall. Hong Kong.1991 11.Cornell educational courses .Poisonous Plants Pyrrolizine alkaloids:Extraction of PAs from materials.heak/pyrol.625htm.1997. 12.EL-Masry S,Korany MA ,Abou-Donia AH. Colorimetric and Spectrophotometric Determination of hydrastis alkaloids in pharmaceutical preparations. J. Pharm. Sci.1980. 69 (5); 597-8. 13.Sakai T. Role of cinchona-alkaloids on the formation of ion-associates between sulphonephthelein dye-stuffs and quarternary ammonium ions.Dept. of Appl. Chem. Archi Institute of Tech. 1247, yachigusa, Yakusa-cho. Tokyo. 2000. 14.Browning DR. Chromatography. Mc. Graw-Hill. London.1969. 15.Pavia DL, Lampman GM, Kriz GS. Organic Laboratory Techniques A micro scale approach. 2nd edn. Saunders Collage Publishers. N Y. 1995.

Discussion:
Thin layer chromatography (tlc) is a very versatile analytical tool used for both qualitative and quantitative investigations. According to Pavia et.al. It can be used to achieve among other things: - establishing that two compounds are identical and to determine the number of components in a mixture [15].The chromatogram suggests that the components of A and C samples are identical with respect to the dye-stuff elution towards the solvent front. Where as sample B at pH 7.23 extracted no quaternary alkaloids, however, the dye stuff alone exhibited similar Rf value to those of dye-stuff liberated by samples C and A. Alstonia boonei is well known for its useful therapeutic importance. Plumiaroidaceae of which Alstonia (50 species), is a member of the sub family includes among other constituents, a vast range of indolic alkaloids some of which are shown in figures I, II and III above. Most of these alkaloids that are quaternary in nature tend to defy the conventional acid-base salt out extraction techniques. Apart from more sophisticated instrumental methods, using HPLC, techniques involving ion exchange resins to mention but these few, the iron-pair complexation and Zn-reduction techniques have shown good promise as quick and reliable processes of detecting the presence of quaternary alkaloids of Alstonia boonei stem bark.

Conclusion:
The present study has suggested that Alstonia boonei popularly used therapeutically in African traditional medicine contains among other secondary metabolites, a significant load of quaternary alkaloids. These alkaloids tend to exhibit a high level of affinity to aqueous acid fraction during their extraction processes, and are generally seen as a serious challenge to Chemists in resource - limited nations and those who are far away into the woods. This technique that has been explored therefore deserves a further attention as it has also the Conflict of interest:-None Source of funding:- Not declared
*Corresponding author:

Oguegbulu, N. Edwin.,

Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Rivers, State Nigeria. Contact no:- 08037124634. .
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