Phytochemical Screening and Preliminary TLC Characterization of Alkaloids in Sabicea brevies Root

INTRODUCTION

Herbal remedies from plants continue to provide a popular alternative for treating known and emerging disease that have defiled many orthodox medical treatments. Herbal medicines are also in great demand in the developing world for primary health care because of their efficiency, safety and lesser side effects. According to World Health Organization (WHO) traditional medicines are relied upon by 60-80% of the world’s population for their primary health care needs (WHO, 2002; Zhang, 2004). Most of these herbal remedies have stood the test of time, particularly for the treatment of allergic, metabolic and cardio-vascular diseases (Igoli et al., 2005). Sabicea brevipes plant belongs to the rubivacea family that has more than 6,500 species out of which 152 are members of the Sabicea genus (Davis et al., 2009). It is an erect or climbing shrub, which is usually 0.6-1.2 m in height. The shrub has a mass of red flowers in the month of July. It is normally found in some part of Africa, Madgascar and America.

There is a need for novel erectile dysfunction treatment drugs, as some of the currently available drugs are frequently associated with lots of side effects. A typical example is with Viagra known for abnormal vision, chest pain, diarrhea, dyspepsia, flushing, headache, hypertension, indigestion, nausea, palpitation, photophobia, priapism and temporary rash with possible severe side effects of intraocular pressure, myocardinal infraction, severe hypotension, stroke, sudden death and ventricular arrahytmias (Akash et al., 2005). There is some evidence suggesting that Sabicea brevipes plant root may help in remedying erectile dysfunction. Sabicea brevipes is a herb used for centuries in Oghe community traditional folk medicine practice as a remedy for erectile dysfunction. Indeed, more than 60 species are used for more than 70 medicinal indications which include sexual weakness (Karou et al., 2011). Maca remains one of the most popular natural remedies in Peru for erectile dysfunction and as an overall male sexual stimulant and libido aid (Oshima et al., 2003). Ashwagandtia, tribulus berries, catuaba (Antunes et al., 2001); Lepidum meyenii walp, daminana, Yohimbe (Pausinystalid yohimbe) (Ernst and Pittler, 1998; Rowland et al., 1997); Muria Puama (Lirisoma ovata), Ginkgo Biloba (Folium ginkgoidis) all contain different kinds of alkaloids that act as vasodilator and androgen precursor. Medical studies have shown that muria puama and catuaba dilates blood vessels and are strong tonic and nervous system fortifies due to the alkaloids present in them that enhance erectile function (Hollman and Katan, 1999; Di Carlo et al., 1999).

It can be said that Sabicea brevipes when probably in central nervous system increase blood flow to the penis. Men with penile circulatory problem should typically get the best result with the root while psychological impotence also fare well using the root product (Reid, 2004; Brown 1995). The overall objective of this study is an assessment of the extracts of Sabice brevipes (susu) plant root samples for selected active ingredients responsible for enhancement of erectile functioning in men and to isolate and conduct preliminary TLC characterization of alkaloid extracted from the roots of Nigeria Sabicea brevipes using selected solvent systems.

MATERIALS AND METHODS

Sample collection: The plant material (Sabicea brevipes root) was collected from Oghe in Ezeagu Local Government Area of Enugu State Nigeria on 10th July 2009 by 11.15 a.m. Presented and defended on September 2011. The herbarilum specimen was identified by Prof. J.C. Okafor of Applied Biology and Biotechnology, Enugu State University of Science and Technology, Enugu State, Nigeria.

Extraction and preparation of plant extract: The root was sun dried for 14 days and pulverized with mechanical grinder. About 42.7 g the powdered plant material was weighed and extracted successively and exhaustively with n-hexane, ethyl acetate and methanol for 4 h, respectively using soxhlet extractor technique. The extracts were concentrated on a reduced pressure to yield a brown-coloured paste (n hexane extract 24.8 mg, ethyl acetate extract 19.4 mg and methanol extract 27.3 mg, respectively). The crude root extracts were later screen qualitatively for the phytochemical constituent utilizing standard methods of analysis (Vishnoi, 1979; Sofowora, 1993; Trease and Evans, 2002).

Phytochemical tests: To about 2.5 cm³ of each extract was added to 10 cm³ of distilled water in a test tube and shaken vigorously with 2 cm³ of olive oil. Fronting which persisted was taken as an evidence for the presence of saponins (Sofowora, 1993). Exactly 2.5 cm³ of each extract was added to 2.5 cm³ of mixed fehling solution A and B in a test tube. The appearance of bluish green precipitate indicates the presence of sugar bonded to saponin as a non-carbohydrate moiety (Sofowora, 1993). Liebemann-Burchard Test for Steriod and Triterpoenoids: To 2 cm³ of each extract was added to acetic acid in a test tube and conc. sulphuric acid (H₂SO₄) was carefully added. Colour development from violet to blue-green indicates the presence of a steroidal ring (Sofowora, 1993). Diluted sulphuric acid was added to each extract in a test tube and boiled for 15 min. Then 10% sodium hydroxide and mixed Fehling’s solution were added. The formation of brick red precipitate indicates the presence of glycoside (Sofowora, 1993). A drop of ferric chloride was added to each extract in a test tube. Then glacial acetic acid and conc. H₂SO₄¯ were added. The development or appearance of blue layer indicates the presence of digitals glycosides (Sofowora, 1993). Born-Traggers Test was used to detect antracenes or anthraquinones. Two 2 cm³ of chloroform was added to each extract in a test tube and allowed to separate. To the chloroform layer was added 10% ammonia solution and vigorously shaken and kept to separate. The formation of brick-red precipitate indicates the presence of antracenes or anthraquinones (Sofowora, 1993). A mixture of 4 cm³ of each extract and 4 cm³ of distilled water was stirred very well in a test tube and 3 drops of ferric chloride added. The occurrence of a blue-black, green or blue-green precipitate indicates the presence of tannins (Trease and Evans, 2002). About 4 cm³ of 10% ammonia solution was added to 4 cm³ of each extract in a test tube and shaken very well. The formation of an emulsion indicates the presence of hydrollisable tannins (Sofowora, 1993). A matchstick was dropped into a 3 cm³ of each extract in a test tube and 2 drops of conc. HCl added. Then the resulting mixture was left undisturbed for 5 min. The development of a dark purple colouration indicates the presence of pseudotannins (Sofowora, 1993). Shinodas test for Flavonoids a small quantity of magnesium chips was dropped into 3 cm³ of each extract in a test tube and 5 drops of conc. HCl added. A pink, orange, or red to purple colouration indicates the presence of flavonoids (Trease and Evans, 2002). Resins was detected by adding 2 cm³ of acetic anhydride to 2 cm³ of each extract in a test tube. Then 2 drops of conc. H₂SO₄ was added to the mixture. The appearance of violet colouration indicates the presence of resins (Sofowora, 1993). The presence of alkaloids was confirmed thus: 2 cm³ of each extract was taken individually into 4 test tubes. To the test-tubes, 2 drops of Dragendorff’s reagent, Mayer’s reagent, Wagners’s reagent and 10% (w/v) tannic acid were added, respectively. Occurrence of orange-red precipitate, appearance of blue-coloured precipitate, observation of a deep brown precipitate and development of cream colouration is a positive tests, respectively (Sofowora, 1993; Trease and Evans, 1978, 1989). Six drops of Ferric chloride was added to a mixture of 2 cm³ of each extract and 2 cm³ of 90% (v/v) ethanol in a test tube. The observation of green colouration indicates the presence of volatile oil (Sofowora, 1993).

Isolation and purification of alkaloids: The roots of the harvested plant sample were cut off, dried and ground using a mechanical grinder. About 180 g of ground plant material was acidified with alcoholic solution of tartaric acid (15 g tartaric acid dissolved in 900 cm³ of 95% ethanol) and warmed to 50°C. This was left in 2000 cm³ conical flask to stand overnight and thereafter filtered. The filtrate was partially evaporated and filtered again to remove fat and protein before the evaporation was completed. The residue was dissolved in boiling 100 cm³ of 95% (v/v) ethanol and the solution cooled overnight in a refrigerator. The alcoholic extract was filtered and the filtrate evaporated to dryness under reduced pressure. The residue was dissolved in 50 cm³ of 2% sulphuric acid (v/v) and the fat extracted with light petroleum ether (40-60°C). The light petroleum ether extract was washed with 200 cm³ of 2% sulphric acid (v/v) and the washing added to the aqueous solution and later neutralized extract was evaporated to dryness and the resulting residue dissolved in 100 cm³ sodium carbonate. The neutralized extract was evaporated to dryness and the resulting residue dissolved in 100 cm³ of absolute ethanol and filtered and the filtrate evaporated to dryness under reduced pressure. The residue was dissolved in 100 cm³ of 1 Mol dm^-3 acetic acid and 50 cm³ of 0.25 mol dm^-3 aqueous solution of lead acetate and warned. The coagulated precipitate was filtered off. The excess lead was removed by cautiously adding in drops dilute sulphuric acid and the lead sulphate filtered off. At this stage non-basic poisons were removed by extracting twice with 30 cm³ petroleum ether. The aqueous liquid was made alkaline with 2 mol dm^-3 sodium hydroxide and the alkaloids except morphine were extracted three times by shaking with 25 cm³ of chloroform. The chloroform extract was later evaporated to dryness in a water bath and the alkaloid weighed and the weight recorded.

For the morphine alkaloid analysis the procedure was thus: the aqueous liquid was acidified with 2 mol dm^-3 of H₂SO₄ and then made alkaline with ammonia and equal volume of methanol (110 cm³) added. The morphine alkaloid was extracted three times with 25 cm³ of chloroform and the extracts evaporated in a water bath and the morphine alkaloid weighed and the weight recorded (Allport, 1951; Dickel et al., 1958; Grinkerich and Safronich, 1983).

TLC analysis for alkaloids: All thin-layer chromatographic analysis was performed using TLC plates pre-coated aluminium oxide F-254 (Type E) with layer thickness of 0.25 mm made in Germany by E. Merck, Darmstadt.

TLC plate (F-254, type E) was used to separate the active compounds present in the extracts (Hashmi et al., 1968). The TLC sheets after development are visualized by spraying with Dragendoff’s reagent.

RESULTS AND DISCUSSION

The result of the phytochemical screening tests conducted on the crude extracts of Sabicea brevipes roots shows the presence of saponins, tannins, glycosides, alkaloids, steroids and triterpenoids as well as volatile oils as shown in Table 1.

The phytochemical test of the crude n-Hexane, ethyl acetate and methanol extracts of Sabicea brevipes roots revealed the presence of steroids, glycosides, alkaloids, saponins, tannins and volatile oil (Table 1). The active principles identified in the n-hexane extract (HE) were steroids, alkaloids (in Wagner’s and Dragendorff’s reagents). Surprisingly, the Mayers and Tannic Acids tests for alkaloids were negative. This observation for alkaloids also occurred in the Ethyl Acetate Extract (EA) and Methanol Extract (ME).

The active ingredients found in the ME were saponins, saponin glycosides, tannins, pseudo tannins, steroids and volatile oils.

Table 1:	Phytochemical constituents of root of Sabicea brevipes

+: Present, -: Absent, HE: N-Hexane extract, EA: Ethyl acetate extract, ME: Methanol extract

The root extracts in the three solvents-HE, EA and ME-show no presence (negative results) for the following active principles: hydrolysable tannins, pseudo tannins, digitalis glycosides, anthracenes, Mayers’ test alkaloids, tannins acid test alkaloid, flavonoids and triterpenoids.

This positive observations help in providing chemotaxonomic evidence for the classification of the species since Sabicea brevipes belongs to the rubiaceea family, which has been reported to contain these compounds (Stary and Storchova, 1991). The identification of these family of compounds further supports claims of the use of the root as a traditional medicine as these compounds have valuable amoebicidal, anti-fungal and anti-inflammatory properties (Opakunle and Jayicoba, 1988). They have stimulating and tonifying effects on the muscles when consumed and this probably accounts for their use in enhancing male potency. Furthermore, the metabolites: alkaloids, steroids, glycosides, saponins and tannins found in the root extracts are known to have curative activity against several pathogens and therefore the plant root is use traditionally for the treatment of various illnesses (Hassan et al., 2004; Usman and Osuji, 2007).

Probably the constituents of Sabicea brevipes from which its male potency enhancing properties emanated are based upon the actions of certain steroidal alkaloid and glycosides. A study has implicated saponin component of plants in enhancing aphrodisiac properties due to its androgen increasing property (Gauthaman et al., 2002). Saponins present in the methanol extract of this plant might have assisted in stimulating an increase in the body natural endogenous testosterone levels by raising the level of Leutinizing Hormones (LH). The LH release normally by the pituitary gland helps to maintain testosterone levels, as LH increases, so does the testesterone (Gauthaman et al., 2002). The increase in testosterone seemed to have translated into the male sexual competence. Furthermore, this study suggests that the aphrodisiac action may be mediated through a change in the blood testosterone level (Yakubu et al., 2005).

Analysis of Sabicea brevipes root using aluminium oxide TLC, eluted with chloroform/ethanol (A) solvent system followed by staining with Dragendorff reagent, showed one orange brown sport with R_F values 0.64 and 0.55 each for morphine alkaloids and non-morphine alkaloids, respectively.

Table 2:	Yield, solvent system composition and R_F values of the various alkaloids

NF: Not found

Organ brown spot (R_F value of 0.84 and 0.34) were observed only for morphine alkaloids using the chloroform/ethanol (B) solvent system. The “solvent system” methanol/ammonia (C) solvent system showed one spot for morphine alkaloids (R_F value 0.55) and also one spot for morphine alkaloids (R_F value 0.55) and also one spot each for non-morphine alkaloids (R_F value 0.27). Solvent system D (methanol/ethyl acetate/ammonia) show two spots for morphine alkaloids (R_F values 0.27 and 0.95) and also two spots for the non-morphine alkaloids (R_F value 0.60 and 0.95). Solvent system E (choroform/metahno/ammonia) gave one spot each for the morphine and non-morphine alkaloids with R_F value of 0.55 and 0.52, respectively. Their R_F values were quite close compared with other solvent systems (Table 2).

Results show that morphine alkaloids contain highest number of spots and percent yield which are 7 sports and 0.433%, respectively; while the non morphine (other) alkaloids had 5 sports and 0.25% yield (Table 2). These findings supported the view that alkaloids are commonly present in Sabicea brevipes (Stary and Storchova, 1991). Therefore further studies are needed to isolate these active compounds and elucidate their chemical structures and highlight their bioefficacies.

CONCLUSION

The results of this study shows that the three extracts of the root of Sabicea brevipes indicates the presence of alkaloids in all the extracts and the presence of saponins, saponin glycosides, tannins and volatile oils only in methanol extracts. While steroids/triterpenoids are only present in n hexane extract.

The results also revealed that morphine alkaloids is the main alkaloids (0.43%) in Sabicea brevipes than non-morphine alkaloids.

The plant parts studied here has also been seen as a potential source of useful drugs. Further studies are going on in order to isolate, identify characteristics and elucidate the structure of the bioactive compounds. Male potency enhancing activities of the plant root for the treatment of erectile dysfunction as claimed by traditional healers are also being comprehensively investigated.