Phytochemical screening of the leaf extracts of Hyptis spicigera plant

The present study reports the screening of phytochemical constituents of the leaf extracts of Hyptis spicigera using hexane, ethylacetate and methanol and the leaf powder of the plant. Qualitative analysis of phytochemical constituents showed the presence of the following secondary metabolites vitannins, carbohydrates, saponins, flavonoids, steroids, alkaloids, quinones, coumarin, terpenoids, resins and cardiac glycosides. The quantitative analysis of total phenolics, alkaloids, saponins, terpenoids and flavonoids carried out using standard protocols revealed the presence of flavonoids (8.82%), saponins (6.23%), terpenoids (16.10%), alkaloids (7.55%) and phenolics (20.75%) respectively. Phenolics showed the highest content (20.75%) while saponins (6.23%) gave the least content. The high content of phenolics in the plant showed that H. spicigera plant may contain antioxidant properties and could be a good source of natural antioxidants. Also, the richness in flavonoids, saponins, alkaloids and terpenoids in this plant can be correlated with its medicinal properties used by traditional herbal healers in Northern Nigeria.


INTRODUCTION
Plants have been the subject of human curiosity and use for thousands of years (Ram et al., 2004) and have played important roles in many countries of the world for centuries by providing food, shelter, clothing, agrochemicals, flavours and fragrances and more importantly, medicines (Gurib-Fakim, 2006).Traditional people have relied on medicinal plants to combat various ailments caused bymicroorganisms such as bacteria, fungi and viruses that infect the body system.Plants have indeed formed the basis of sophisticated traditional medicine systems which will continue to provide mankind with new remedies for all forms of ailments (Gurib-Fakim, 2006).
Bioactive natural products have enormous economic importance as specialty chemicals as they can be used as drugs, lead compounds, biological or pharmaceutical tools, feed stock products, excipients and nutraceuticals (Pieters and Vlietinck, 2005).In recent times, focus on plant research has increased all over the world and a large body of evidence and knowledge has accumulated in the literature to show immense potential of medicinal plants used in various medical, pharmaceutical, cosmetic, agrochemical applications.An advantage of natural bioactive molecule is that they have a milder side effect on the body in comparison to chemically synthesized drugs (Badisa et al., 2003).With the increasing acceptance of herbal medicines as alternative form of health care delivery, the screening of medicinal plants for bioactive compounds is imperative (Masoko et al., 2005;Cowan, 1999).
Hyptis spicigera belongs to the family Lamiaceae and is commonly known as Black beniseed, or Black sesame.It is an erect aromatic herb, up to 1 m in height, with a terminal inflorescence in which the seeds are packed in quadruplets or more in the flowers.The plant is found around Senegal to western Cameroon, possibly native to Brazil, now widely naturalized in tropical Africa and Asia as well as Nigeria.It grows naturally and commonly as a weed.It prefers roadsides, waste places, cultivated places and often damp places (Burkill, 1995).Generally, the whole plant is used in traditional stores to protect cowpea against damage by Callosobruchus species (Lambert et al., 1985).The Bajju and Atyapp people of Kaduna state, northern Nigeria, make use of the inflorescence (where the seeds are packed) to cure headaches by sniffing it and also crushing the leaves and applying to the head to relieve head colds and headaches (Dalziel, 1937).
This paper reports the phyto constituents of the leaf extracts of H. spicigera and their potential medicinal applications.

Collection of the plant
About 500 g of the leaf part of H. spicigera was collected in Basawa Village, Zaria, Kaduna state, Nigeria on the 26 th November, 2013.It was taxonomically identified and authenticated by Mallam U. S. Gallah of the Herbarium Section, Department of Biological Science, Ahmadu Bello University Zaria, Nigeria, and a sample Voucher No.528 was deposited at the Herbarium section of the Department of Biological sciences.

Extraction and isolation
The plant was dried in the shade for 14 days and pulverized to powder using pestle and mortar in the Laboratory.Approximately 450 g of the powdered plant material was macerated sequentially with hexane (1 L), ethyl acetate (1 L) and methanol (1 L) at room temperature (27°C) and concentrated in vacuo to afford the various crude extracts which were stored in the refrigerator (4°C) until needed for further analysis.

Salkowski reaction test for phytosterols
To 0.5 ml each of the extracts in a test tube was added 1.0 ml of concentrated H2SO4 (conc.)from the sides of the test tube and then 1.0 ml chloroform.Appearance of reddish brown colour in chloroform layer indicates the presence of phytosterols.

Liebermann-Burchard's test for triterpenoids
Extracts were treated with few drops of acetic anhydride, boil and cool.Conc.sulfuric acid was added from the sides of the test tubes which showed a brown ring at the junction of two layers, and formation of deep red color indicated the presence of triterpenoids.

Foam test for saponins
Small amount (0.1 g) of the extracts were taken in test tubes with little quantity (1.0 ml) of water and shaken vigorously.Appearance of foam persisting for 10 min indicated presence of saponins.

Dragendroff's test for alkaloids
About 0.5 g each of hexane, ethyl acetate and methanol extracts were dissolved in 1.0 ml chloroform and evaporated.The residue was acidified by adding few drops of Dragendroff's reagent (Potassium bismuth iodide).Appearance of orange red precipitate indicated presence of alkaloids.

Molisch's test for carbohydrates
About 0.5 g each of the extracts was mixed with Molisch reagent, and then added H2SO4 conc.along the sides of the test tube to form layers. Appearance of reddish violet ring the interference indicated the presence of carbohydrates.

Lead acetate test for flavonoids
To 0.1 g each of the extracts were dissolved in ethanol and few drops of 10% lead acetate solution were added.Appearance of yellow precipitate indicated presence of flavonoids.

Legal's test for lactones
To 0.1 g each of the extracts1.0ml sodium nitroprusside and1.0 ml pyridine were added in test-tubes.The mixtures were treated with 0.01 moldm -3 NaOH.Appearance of deep red colour indicated the presence of lactones.

Ferric chloride test for phenolic compounds and tannins
About 2.0 ml of each extract was measured in a test tube and 0.01 mol dm -3 Ferric chloride solution was added drop by drop.Appearance of bluish black precipitate indicated presence of phenolic compounds and tannins.

Ninhydrin test for proteins
Few drops of ninhydrin were added to the extracts.Appearance of blue colour indicated presence of amino acid.Proteins may rarely give positive result with this test.

Keller-Killiani test for glycosides
About 1 ml of glacial acetic acid, few drops of 0.01 mol dm -3 Ferric chloride solution and H2SO4 (Conc) slowly through the sides of the test tube) were added to the extracts.Appearance of reddish brown ring at the junction of the liquids indicated the presence of deoxysugars.

Determination of total phenolic compound (TPC)
Total phenolic content of the hexane, ethylacetate and methanolic extracts was determined by standard method (Makkar et al., 1993) with little modifications, using tannic acid as a standard phenolic compound.The extracts were diluted with distilled water to a known concentration in order to obtain the readings within the standard curve range of 0.0 to 600 μg of tannic acid/ml.250 μl of diluted extract or tannic acid solution was mixed with 1 ml of distilled water in a test tube followed by the addition of 250 μl of Folin-Ciocalteu reagent.The samples were mixed well and then allowed for 5 min at room temperature in order to allow complete reaction with the Folin-Ciocalteu reagent.Thereafter, 2.5 ml of 7% sodium carbonate aqueous solution was added and the final volume was made up to 6.0 ml with distilled water.The absorbance of the resulting blue colour solution was measured at 760 nm using spectrophotometer after incubating the samples for 90 min.All the experiment was conducted in three replicates.

Determination of alkaloids
About 5.0 g of the dried powdered plant was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added.The mixture was covered and allowed to stand for 4 h.This was filtered and the extract concentrated on a water bath to one-quarter of the original volume.Concentrated ammonium hydroxide solution was added dropwise to the extract until the precipitation was complete.The solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide and finally filtered, dried, weighed and the percentage alkaloid was calculated (Harborne, 1998).

Determination of total terpenoids
About 2 g of the plantleaf powder was weighed and soaked in 50 ml of 95% ethanol for 24 h.The extract was filtered and the filtrate extracted with petroleum ether (60 to 80°C) and concentrated to dryness.The dried ether extract was treated as total terpenoids (Ferguson, 1956).

Determination of saponins
About 15 g of each sample was placed into a conical flask and 100 ml of 20% aqueous ethanol was added.The samples were heated over a hot water bath for 4 h with continuous stirring at 55°C.The mixture was filtered and the residue re-extracted with another 200 ml and 20% ethanol.The combined extracts were reduced to 40 ml Ladan et al. 85 over water bath at 90°C.The concentrate was transferred into a 250 ml separating funnel and 20 ml of diethyl ether was added and shaken vigorously.The aqueous layer was recovered while the ether layer was discarded.This purification process was repeated and 60 ml of n-butanol was added.The combined n-butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride.The remaining solution was heated in a water bath.After evaporation the samples were dried in the oven to a constant weight and the percentage saponin was calculated (Obdoni and Ochuko, 2001).

Determination of flavonoids
About 5.0 g of the plant sample was weighed and extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature.The whole solution was filtered through whattman filter paper No 41.The filtrate was evaporated into dryness over a water bath and weighed to a constant weight.The percentage flavonoids was then calculated (Soni and Sosa, 2013).

RESULTS AND DISCUSSION
The extracts of the leaves of H. spicigera were screened for the presence ofthe following secondary metabolites: alkaloids, glycosides, flavonoids, carbohydrates tannins, steroids, terpenoids and resins, coumarins, saponins and quinines.The results of the phytochemical screening showed the presence of all the secondary metabolites analyzed in ethylacetate and methanol extracts while hexane extract showed only the presence of alkaloids, glycosides, carbohydrate and resins.Other secondary metabolites such as flavonoids, tannins, steroids, terpenoids, coumarins, saponins and quinones were absent in the hexane extract.
The phytochemical content was found to be similar to that obtained by other authors (Onayade et al., 1991) with different extracts revealing the different partitioning abilities of the different solvents used.The presence of these phytochemicals in all the extracts is quite instructive as this lends credence of the use of the plant for medicinal purposes.A lot of plants contain non-toxic glycosides that can be hydrolyzed to give phenolic compounds that are toxic to microbial pathogens (Abaoba and Efuwape, 2001).The saponin content in the ethylacetate and methanol extracts were found to be present in these extracts, respectively.Saponins possess the property of precipitating and coagulating red blood cells (Sodipo et al., 1991).It also foamed in aqueous solution and has hemolytic effect and can also bind on cholesterol sites.These properties make saponins present in the plant to exhibit medicinal properties (Sodipo et al., 1991) and this therefore supports the findings in this present study that extracts of the plants may be useful in chemotherapy of mycotic infections which the antimicrobial studies revealed (Ladan et al., 2009).Alkaloids were found present in hexane, ethylacetate and methanol extracts and this can be corroborated with literature reports which indicate that naturally occurring alkaloids and their synthetic derivatives  (Okwu and Okwu, 2004).They exhibit marked physiological activity when administered to animals.Classes of alkaloids are among the major powerful poisons known and despite being poisonous, some of the alkaloids are known to be useful in correcting renal disorders (Konkwara, 1979).The use of some plants for medicinal purpose, in the traditional treatment of diseases is due to the presence of flavonoids and saponins (Zwadyk, 1992;Othira et al., 2009), hence the use of H. spicigera for the treatment of diarrhea, dysentery, colds and several other diseases by local herbalists or traditional healers is not surprising.The presence of flavonoid was evident in methanol and ethylacetate extracts, flavonoid containing plants have been used as diuretic, laxative, emollient and poultice (Baba-Mousa et al., 1999) therefore; the use of H. spicigera rich in saponins and other Hyptis species in traditional medicine lent credence to the medicinal potentials of the plant.Tannins in some medicinal plants have been found to be responsible for the antiviral and antibacterial activities exhibited by such plants (De-Ruiz et al., 2001;Elegani et al., 2002).Therefore, H. spicigera with high tannin content in ethylacetate and methanol extracts could probably be a source of phytochemicals for the treatment of bacterial infections.Phenolic compounds like tannins present in plant cells are inhibitors of many enzymes (proteolytic and hydrolytic) used by plant pathogens.Other compounds such as saponins have antifungal properties (Abaoba and Efuwape, 2001;Mohanta et al., 2007).Therefore, these phytochemicals detected in this study may be responsible for the antimicrobial potency of the leaf extracts of Hyptis spicigera and also lend credence to the claims of traditional application of the plant as remedies for various ailments.

Quantitative phytochemical analysis
Results of the quantitative analysis data of the plant material revealed significant levels of phytochemical constituents present in the leaf as evident in the qualitative analysis data (Table 1).Phenolic content (20.75%) is the highest followed by terpenoids content (16.10%) while flavonoid (8.82%), saponins (6.23%) and alkaloids (7.55%) followed respectively.Subhashini et al. (2013) and Soni and Sosa (2013) have reported various phyto constituents in the leaves of Ecbolium viride (Forks) Merrill plant and the methanolic and ethyl acetate extracts of the leaves of Anogeissus leiocarpus and found the following values: terpenoids (0.3034 w/w), saponins (0.1100 w/w), alkaloids (0.1340 w/w), flavonoids (0.0884 w/w) and phenols (0.03045) for the E. viride (Forks) while alkaloids (152.0 ± 0.1 mg/g ), phenolics 1294.81±3.0 mg/g), flavonoids (330.7 ± 3.0 mg/g) in the methanol extract and alkaloids (80.20 ± 0.0 mg/g), phenolics (616.5 ± 4.4 mg/g), flavonoids (202.5 ± 4.0 mg/g) in the ethyl acetate extract of the A. leiocarpus plant have been reported.The quantitative values of these metabolites reported in the leaf part of H. spicigeraare higher (Table 2) than those reported for E. viride (Forks) Merrill and A. leiocarpus plants.Phenolic compounds are one of the most important constituents of plant secondary metabolites with marked physiological properties.
The phyto constituents found in the plant may be responsible for its biological properties such as antioxidative, anti-inflammation, anti-carcinogenic, antihypertensive, anti-diabetic, anti-cancer, cardiovascular protection and improvement of endothelial function (Han et al., 2007).Several studies have described the antioxidant properties of different parts of various medicinal plants which are rich in phenolic compounds (Brown and Evans, 1998;Krings and Berger, 2001;Malencic et al., 2007).Natural anti-oxidants mainly come from plants in the form of phenolic compounds, such as flavonoids, phenolic acids, tocopherolsetc (Ali et al., 2008) and used for the treatment of degenerative diseases.The antioxidative properties of flavonoids are due to several different mechanisms, such as scavenging of free radicals, chelation of metal ions, such as iron and copper and inhibition of enzymes responsible for free radical generation (Akinmoladun et al., 2007;Benavente-Garcia et al., 1997).This plant (H.spicigera) will provide the natural anti-oxidant needed to enhance good living by scavenging free radicals that cause ill health in humans.

Conclusion
Phytochemical screening of the leaf part of H. spicigera revealed the presence of tannins, carbohydrates, saponins, flavonoids, steroids, alkaloids, quinones, coumarin, terpenoids, resins and cardiac glycosides which are important secondary metabolites.The richness of the plant in phenolic contents and other secondary metabolites affirmed its medicinal efficacy and potentials.
The finding from this study therefore suggests that the leaf could be a potential source of natural anti-oxidant that could have great importance as therapeutic agents in preventing or slowing ageing associated with oxidative stress and related degenerative diseases.It is recommended that further investigation on the isolation and characterization of the bioactive constituents of the leaf leading to structural elucidation is necessary.

Table 1 .
Qualitative phytochemical analysis of the leaf extracts of Hyptis spicigera.

Table 2 .
Quantitative analysis of the leaf part of Hyptisspicigera.