Introduction 

Plant seeds are common sources of oils which may have nutritional, industrial and pharmaceutical importances¹. Suitability of an oil for nutritional purpose is however dependent to a considerable extent on its fatty acid composition. Generally, oils from different sources differ in their fatty acid compositions and most of the oils from single sources have been found to be unsuitable for all purposes. The patterns of fatty acid variation in plant seed oils have proven to be useful tools in taxonomic and phytogenetic studies². Large number of non-conventional plants have been surveyed in search for new source of oils³. Alstonia scholaris, one of such non-conventional plants belongs to the family Apocynaceae, is widely found in the Indian subcontinent and has various folk medicinal uses. This plant is known for its healing and curative properties for a long time and it is an excellent and popular substitute for cinchona and quinine for the treatment of intermittent and remittent fevers⁴. The bark is regarded as a bitter tonic and a mild febrifuge, possessing astringent, antihelmintic and galactogouge properties⁵. It is also reported to be employed in heart diseases, asthma, chronic diarrhea and to stop bleeding of wounds⁵. It is also administered in leprosy, dyspepsia, skin diseases and even in cancer like conditions⁵. Decoction of young leaves is used traditionally in Malaysia for Beri-beri, congestion of liver and dropsy⁵. No reports are presently available on Alstonia scholaris seed oil and its use for edible as well as for medicinal purpose.

The present study deals with some investigations for characterization of fatty acids and their composition of the seed oil and tracing existence of bioactive ingredients in the seeds of Alstonia scholaris. Such study will be potential for possible future domestication and safe  use for human consumption⁶. Bioactivity studies will help to realize the potential of the seed oil as medicine. Furthermore, these informations may also be used for taxonomic and evolutionary studies.

Experimental  Section

Materials And Methods

Plant material and chemicals:

Fresh mature seeds of Alstonia scholaris were collected from the Burdwan Divisional Forest Department, Burdwan, West Bengal, India and authenticated by Prof. A. Mukherjee, Department of Botany, University of Burdwan, Burdwan, West Bengal, India. Voucher specimen Burdwan, Mita 199 has been deposited at the herbarium of the Department of Botany at the University of Burdwan, Burdwan, bearing acronym BURD.

Standard fatty acids used in the experiment were purchased from Sigma Chemical Co., USA. All reagents and chemicals used in this investigation were of analytical grades.

Isolation and Characterisation of Seed oil

The seeds were taken out of the pod and dried in air. The finely powdered air dried seeds (405 gms) were extracted with  hexane in a soxhlet for 60 hrs and after complete removal  of the solvent under vacuum, seed oil was obtained. The total oil was weighed and stored under nitrogen at 4^oC for further analysis. The chemical analysis of the seed oil (including acid, iodine and saponification values) were performed according to the methods of Association of Official Analytical Chemists⁷ and the results have been placed in Table 1.

Extraction and identification of fatty acids:

The extraction of fatty acids and its methyl ester preparation were performed according to the method described by G.H. Wikfors et. al⁸. Methyl esters of fatty acid mixture of the seed oil was purified by preparative TLC using hexane: ethyl acetate (1:1) as chromatographic solvent and fatty acid methyl ester band was eluted with chloroform (Merck, India) and stored in refrigerator for further analysis.

GC analysis

Gas chromatographic analysis of the purified methyl ester of fatty acids of seed oil of A. scholaris was carried out with the aid of a Hewlett Packard (HP; Palo Alto, CA, USA Model, Agilent 6890 series plus) instrument fitted with HP-5 capillary column (30m long; 0.25 mm i.d) using a    flame ionization detector (FID), the temperatures of the injection and detector ports were set at 250^oC. The oven temperature was initially 160^oC (held for 2 min), then raised at 3^o/min to 220^oC (held for 15 mins). The carrier gas was nitrogen (flow rate of 20ml/min); volume injected 1 ml; split ratio,1:20. Peaks were identified by comparision of their retention times with that of standard fatty acids methyl esters. The percentage composition of the samples were computed from the GC peak areas. The results obtained were placed in Table 2.

Inhibitory test with seed oil

Inhibitory test was performed with the total seed oil, saponified  seed oil and acidic and neutral parts of the seed oil (after separating it into acidic, basic and neutral fractions).

Bacterial organisms (a few gram positive and gram negative organisms ) taken in the experiment were Bacillus subtillis, Eschericia coli, Salmonella sp. and Bacillus sp. Solutions of total seed oil, saponified seed oil, acidic neutral fractions of the seed oil (2mg/mL in each case) were given separately to observe its activity against the aforementioned micro-organisms. The cup bore method⁹ was used to perform susceptibility test¹⁰. The zones of inhibition were estimated visually and the data were placed in Table 3.

Results

Oil (pale yellow in colour) content of this seed was found to be 239.5g/kg (weight per kg dry matter of seeds). The characteristics of the seed oil of A. scholaris is given in Table 1.

Table 1: Characteristics of the seed oil.

The acid value is indicative of the amount of free fatty acids present in the oil. From the GC analysis of the methyl esters of fatty acids present in the oil of matured seeds of A. scholaris, indicates four fatty acids were present in the oil. They were identified and quantified ( Table 2 ) representing 100% of the fatty acids.

Table 2: Fatty acid compositions of the seed oil of Alstonia scholaris.

Results of the inhibitory test performed with the seed oil of A.Scholaris, saponified oil, acidic and neutral fractions of the seed oil as tabulated in Table 3 indicate that the seed oil, saponified seed oil and acidic fraction of oil has profound activity against the test microorganisms.

Table 3 : Antibacterial activity obtained from Inhibitory test

Cup diameter = 8 mm; Sample used = 0.1 ml/cup

Medium used for assay: Nutrient agar

Solvent as control: Hexane

Discussion

Oleic acid (C18:1 n-9) was the principal unsaturated fatty acid (65.66%), followed by linoleic acid (C18:2 n-6) as the second main unsaturated fatty acid (12.5%). Palmitic acid (C16:0) and stearic acid (C18:0) were the other saturated fatty acids present their in. Their relative amounts being 13.77% and 8.42%. The proportions of unsaturated and saturated fatty acids are 77.81% and 22.19% respectively.

The oil of this seed is rich in two unsaturated fatty acids i.e., oleic and linoleic acid. Both of these acids are important from the nutritional point of view as well as for oil stability. It is well known fact that dietary fats rich in linoleic acid prevent cardiovascular disorders like coronary heart diseases and high blood pressure. Its derivatives also serve as structural components of the plasma membrane and as precursors of some metabolic regulatory compounds¹¹. The result of the present investigation indicates that A. scholaris seed oil may be a good source of essential fatty acids and thus it may be nutritionally valuable. Moreover, high content of linoleic acid may render the oil interesting for cosmetic industry.

As evident from the inhibition zones from Table 3 it is clear that the acidic fraction of the seed oil showed highest activity against the test organisms.

Conclusion

The hexane extracted oil from the seed of A. scholaris contains four fatty acids of which two are very essential unsaturated fatty acids (oleic acid and linoleic acid). Moreover, the total oil and the acidic part of the oil showed profound activity against E. coli, Salmonella sp., Bacillus subtilis, Bacillus sp. Thus, it can be concluded that the oil may be important from nutritional as well as medicinal point of view after proper toxicological study.

Acknowledgement

Authors are grateful to University Grants Commission, New Delhi, India for financial assistances.

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