Characterization of Annona muricata stem methanol extract by gas chromatography–mass spectrometry analysis

 

Okoro Ebere Kelechi, Amaku Friday James, Okoro Innocent Ajah

Department of Chemistry, Michael Okpara University of Agriculture, Umudike, Nigeria

 

ABSTRACT:

Annona muricata extract are extensively used as traditional remedy for a variety of illnesses like cancer, diabetes, hypertension and malaria. Methanol extract of Annona muricata (soursop) stem were analyzed using gas chromatography–mass spectrometry  method.  The result obtained from the GC-MS analysis of methanol extract of Annona muricata stem led to identification of ten (10) compounds in which 1-(4-hydroxy-6-methyl-2-pyrimidinyl)-2-aziridinone, hexadecanoic acid (palmitic acid) and 9-octadecanoic acid(oleic acid) are the major component reported with significant bioactive impact. The components were identified by comparing their retention indices and mass spectra fragmentation patterns with those stored in the National Institute of Standards and Technology (NIST) library.

 

 

INTRODUCTION:

A large number of plants possess antibiotic properties and are used extensively by different tribes  worldwide. Rapid explosion in human population has made it almost impossible for modern health facilities to meet health demands all over the world, thus putting more demands on the use of natural herbal health remedies. The use of, and search for, drugs and dietary supplements derived from plants have accelerated in recent years. Pharmacologists, microbiologists, botanists, and natural-products chemists are combing the earth for phytochemicals  that could be developed for treatment of various diseases. In fact, according to the WHO, approximately 25% of modern drugs used in the United States have been derived from plants ^[1].Among the 120 active compounds currently isolated from the higher plants and widely used in modern medicine today, 80 percent show a positive correlation between their modern therapeutic use and the traditional use of the plants from which they are derived ^[2].

More than two thirds of the world's plant species - at least 35,000 of which are estimated to have medicinal value - come from the developing countries. In many medicinal and aromatic plants (MAPs) significant variations of plants characteristics have been ascertained with varying soil traits, and the selective recovery and subsequent release from food.

 

Annona muricata L (Soursop) is a widespread small tree and it is native to Central America ^[3]. It is a typical tropical tree with heart shaped edible fruits and widely distributed in most tropical countries. In Nigeria, it is known as Ebo in Yoruba, Shawanshop in Igbo and Tuwon biri in Hausa. All parts of A. muricata (soursop) tree are used in natural medicine in the tropics including the twigs, leaf, root, fruit and seeds. Generally, the fruit and fruit juice are taken to eliminate worms and parasites, cool fever, increase mother’s milk after child birth, and as an astringent for diarrhea and dysentery ^[4].The twigs and leaf are considered sedative and antispasmodic ^[4]. Previous reports have demonstrated that the leaf, twigs, root, stem and fruit seed extracts of A. muricata (soursop) have several biological activities such as anti-bacterial ^[5], antifungal ^[6] and anti-malarial ^[7].The leaf, stem, root and seed extract of Annona muricata showed antibacterial activity against numerous pathogens ^[8]. The leaves, roots and seeds of Annona muricata  demonstrated insecticidal properties, with the seeds demonstrating strong insecticidal activity ^[9]. A. muricata leaf extract exhibit a broad spectrum of activity against a panel of bacteria like B. subtilis, Staph aureus, K. pneumonia, P. vulgaris,  responsible for common bacterial diseases like pneumonia, diarrhea, UTIs and skin infections ^[10]. Aqueous extracts of A.muricata exhibit antibacterial effect against S. Aureus and Vibrio cholerae. The ethanolic extract of the  leaf showed highest antibacterial activity towards Pseudomonas aeruginosa and Staphylococcus aureus ^[11].

 

MATERIALS AND METHODS:

Sample collection, preparation and preservation

The study was carried out using young stems harvested from a soursop tree in Emekuku.  The young stems were harvested from different branches of the tree, stripped of their leaves and then cut into small for further analysis. The collected samples (stem) were air-dried properly for three weeks, milled and sieved into fine powder using a 2mm mesh size sieve. The powders were then stored in properly labeled air-tight containers until required for analysis.

 

Sample extraction and purification

All chemicals used were of analytical grade and were used without further purification. They were purchased from BDH Chemicals, London.. One hundred grams (100 g) of the powdered stem sample was introduced into a conical flask containing 250 ml of 98% methanol. The mixture was thoroughly shaken and allowed to stand for 48hours.The mixture were filtered using whatman No.1 filter paper. The residues were rinsed with ethanol, filtered again and the two filtrates combined for concentration. The filtrates were concentrated to near-dryness in a water bath at 60after which the remaining solvent was allowed to evaporate at room temperature. The crude extract were stored properly and labeled for purification. Separation and purification was carried out using column chromatography method. 11g of stem crude extract were weighed into labeled beakers and dissolved with a little amount of petroleum ether. Silica gel was then added to the extract, a little at a time and stirred until a free-flowing slurry is achieved. The stem extract slurries were then loaded unto already packed columns and elution is commenced. A three-solvent system (petroleum ether, acetone and methanol) was used in the elution process in mixtures of increasing order of polarity. Fractions were collected in 50ml beakers at a volume interval of 20ml/ fraction and at the end, 30 fractions of the stem extract were collected.

 

TLC analysis

All the fractions collected were subjected to TLC analysis to see if there are any pure fraction(s). The plates were coated with silica gel and allowed to dry at room temperature. The fractions from column chromatography were then spotted on the dried silica gel-coated plates using a micro pipette. Solvent mixtures of varying polarities were used to run the TLC which gave different R_f values for each fraction. Table 1.0, below shows the R_f values of some stem fractions in acetone and petroleum ether mixture of 70 %: 30 % v/v.  The stem extract fractions, ST₈, ST₉ and ST₁₀ were pooled together to form a composite ethanol extract of the soursop stem and was sent for chemical analysis using GC-MS. 10ml of each fraction was transferred into appropriately labeled sample bottle, carefully sealed and sent to National Research Institute of Chemical Technology (NARICT), Zaira, Kaduna state, Nigeria for GC-MS analysis.

 

GC-MS analysis

Characterization of the plant extract was performed using GC-MS QP2010 Plus (Shimadzu, Japan) with Thermal Desorption System, TD 20 coupled with Mass Spectroscopy (Shimadzu). The ionization voltage was 70eV. Gas Chromatography was conducted in the temperature programming mode with a Restek column (0.25 mm, 60m, XTI-5). The initial column temperature was 80^oC for 1min, and then increased linearly at 70oC min-1 to 220^oC, held for 3 min followed by linear increased temperature 10^oC min-1 to 290^oC for 10 min. The temperature of the injection port was 290^oC and the GC-MS interface was maintained at 290^oC. The sample was introduced via an all-glass injector working in the split mode, with helium carrier gas low rate of 1.2 ml min^-1.

 

 

Result and discussion

Calculated R_f values of some fractions of Annona muricata  stem extract are presented in Table 1. Figure 1 shows thegas chromatogram of  methanol extract of A. muricata  stem.. Table 2 shows the results obtained from the GC-MS analysis. The mass spectra of methanol extract of A. muricata  stem are presented in Figures 2 – 11.

 

Table 1:     Calculated R_f values of some fractions of stem extract using petroluem ether: acetone mixture (30:70) v/v.

 

 

Figure 1:    Gas chromatogram of methanol extract of A. muricata stem

 

 

Figure 2: Mass spectrum of nonane

 

 

 

Figure 3: Mass spectrum of 2-methyl decane

 

 

 

Figure 4: Mass spectrum of 2,7-dimethyl octane

 

 

 

Figure 5: Mass spectrum of 2-methyl nonane

 

 

Figure 6: Mass spectrum of sulphurous acid,2-propylundecyl ester

 

 

 

Figure 7: Mass spectrum of 3,7,11-trimethyl -1,6,10-dodecatrien-3-ol

 

 

Figure 8: Mass spectrum of 2,3,3-trimethyl octane

 

 

 

Figure 9: Mass spectrum of 1-(4-hydroxy-6-methyl-2-pyrimidinyl)-2-aziridinone

 

 

 

Figure 10: Mass spectrum of hexadecanoic acid (palmitic acid)

 

 

Figure 11: Mass spectrum 9-octadecanoic acid (oleic acid)

 

 

 

 

           Figure 12:  Nonane

 

 

Figure 13: 2-methyl decane

 

 

Figure 14:  2,7-dimethyl octane

 

 

Figure 15: 2-methyl nonane

 

 

 

Figure 16: Sulphurous acid,2-propylundecyl ester

 

 

 

Figure 17:   3,7,11-trimethyl-1,6,10-dodecatrien-3-ol

 

 

 

 

 

Figure 18: 2,3,3-trimethyl octane     

 

 

 

Figure 19: 1-(4-hydroxy-6-methyl-2-pyrimidinyl)-2-aziridinone

 

 

 

Figure 20: Hexadecanoic acid (palmitic acid)                  

 

 

 

 

 

Figure 21: 9-octadecanoic acid (oleic acid)

 

 

 

 

 

 

 

 

 

Table 2:     Organic compounds identified using GC-MS in the methanol stem extract of A. muricata (Soursop).

Peak No.	Compound name	Molecular formular	Molecular mass	% Content	Retention time(min)	Nature of compound Hydrocarbon
1	Nonane	C9H20	128	8.28	3.327
2	2-methyl decane	C11H24	156	9.48	4.393	Hydrocarbon
3	2,7-dimethyl octane	C10H22	142	3.96	5.681	Hydrocarbon
4	2-methyl nonane	C10H22	142	0.84	7.059	Hydrocarbon
5	Sulphurous  acid,2-propylundecyl ester	C14H30O3S	278	0.57	8.438	Sulphurous acid ester
6	3,7,11-trimethyl-1,6,10-dodecatrien-3-ol	C15H26O	222	0.88	12.141	Terpene
7	2,3,3-trimethyl octane	C11H24	156	1.10	13.452	Hydrocarbon
8	1-(4-hydroxy-6-methyl-2-pyrimidinyl)-2-aziridinone	C7H7N3O2	165	0.90	15.293	Alkaloid
9	Hexadecanoic acid(palmitic acid)	C16H32O2	256	17.08	18.674	Fatty acid
10	9-octadecanoic acid(oleic acid)	C18H34O2	282	56.91	21.410	Fatty acid

 

 

3 fractions of the stem extract (ST₈, ST₉, and ST₁₀) gave single spots of R_f values 0.50, 0.35 and 0.49 respectively. Table 2 shows the results obtained from the GC-MS analysis. The methanol extract of the stem of A. muricata (soursop) showed ten peaks as observed in the gas chromatogram of the extract in Figure 1, these peaks indicate the presence of ten compounds in the stem extract. Figure 12- 21 shows the molecular formulae of compounds in the stem extracts. For the stem extract, the compounds comprises of alkanes (23.66%), sulphurous acid ester (0.57%), sesquiterpene(0.88%), alkaloid (0.90%), palmitic acid(17.08%) and oleic acid (56.91%). Fatty acids especially palmitic and oleic acids have been employed for their pharmacological and medicinal effects.  Palmitic acid is required for biosynthesis of lung lecithin, which is related to fetal maturation^[12]. Radiochromatogram showed high incorporation of palmitate into lecithin by fetal lung. High content of palmitic acid in palm oil in Nigerian meals can partly be related to low incidence of respiratory distress ^[12]. In the search for anticancer agents with fewer side effects, palmitic acid and oleic acid has been shown experimentally to markedly suppress the granulosa cell survival and dose-dependent manner via apoptosis ^{[13, 14]}. The high fatty acid content of the seed and stem ethanolic extracts may partly account for the documented anticancer properties of A. muricata .

 

The stem extracts of A. muricata is used extensively as insecticides or pesticides. According to Kulenkampff, mono alpha carboxylic acids especially palmitic and oleic acids are used to formulate arthropodicidal solutions effective against insects and arachnid pests as well as fungicidal and moss killing compositions ^[15]. The high palmitic acid and oleic acid content of the methanol stem extracts can be seen from the GC-MS result as shown in Table 2. This is most likely responsible for the effective use of A. muricata (soursop) stem extracts as an insecticide and/or pesticide as shown by ^[16]. Compound 6 from the stem extract is a sesquiterpene as seen in Figure 17, this is a sesquiterpene  known as nerolidol. The activities of nerolidol include its application as a food-flavoring agent and currently under test as a skin penetration enhancer for the transdermal delivery of therapeutic drugs. It was evaluated against Leishmania species and it was found to inhibit the growth of Leishmania amazonensis, L. braziliensis, and L. chagasi promastigotes and L. amazonensis amastigotes ^[17] .

 

The alkaloid present in the stem extract as seen from the GC-MS result is known as Elastopar as seen in Figure 19. The presence of an aziridinium moiety in the compound may mean that compound can be used as an anticancer agent as it is likely to have the same reaction mechanism with alkylating anticancer agents ^[18]. This compound may also be partly responsible for the anticancer properties of methanol stem extract of A. muricata.  Compound 5 of the methanol stem extract (Table 2) is a sulphurous acid ester (Fig. 16). This is an  organosulphates derived from alcohol and sulphuric acid. It is an active ingredient in detergents, shampoos and conditioners. It can also be found in other household products such as toothpastes, antacids, cosmetics and foods ^[19]. According to Gunther and co, sulphurous acid esters especially the open chain alkyl sulphites, show very high efficiency as antioxidants.

           

CONCLUSION:

The methanol extracts of Annona muricata stem (soursop) has been shown to contain quite a number of organic compounds that are of pharmacological importance. These include alkaloids, fatty acids, esters and terpenoids. The GC-MS analysis of pooled stem extract with three similar R_f values,  0.500, 0.350 and 0.490 indicated the presence of ten compounds.  Different parts of this plant are extensively used as traditional remedy for a variety of illnesses like cancer, diabetes, hypertension and malaria.

 

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[14]        Zhang L, Ji J, Zhu XY, Wu YY, Yu H, Zhang B, Li XL and Sun XZ. Palmitic acid induces apoptosis in human hepatoma cell line, HepG2 cells. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 26 (6); 2004: 671- 676.

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Received on 28.07.2016       Modified on 12.08.2016

Accepted on 30.08.2016      ©A&V Publications All right reserved