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QUALITATIVE ANALYSIS OF PHYTOCHEMICAL COMPOUNDS IN OCIMUM BASILICUM GROWN IN QATAR


Ameena Fakhroo1* and Lakshmaiah Sreerama1, 2
1. Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar
2. Department of Chemistry and Biochemistry, St Cloud State University, St Cloud, MN, USA

ABSTRACT

Evaluation of the chemical constituents of Ocimum bacilicum (locally known as Mashmoom) adopted to grow in the desert environment of the Gulf region is limited.  Accordingly, the purpose of this study was to qualitatively evaluate the complete composition of phytochemical compounds present in this plant. Both methanolic and aqueous extracts of Ocimum basilicum grown in Qatar were prepared and tested for various phytochemical present in these extracts using qualitative tests. The analysis revealed the presences of glycosides, tannins, terpenoids, phenols, and phlobatatannins in both extracts.  In addition, the methanolic extracts also contained alkaloids, flavonoids, saponinsand anthraquinones.  On the other hand, neither extracts contained proteins or anthrocyanins. Qualitative tests reveal the presence of various phytochemicals (listed above). The methanolic extracts, in particular, reveal the presence of possibly new or modified anthraquinones and it may be unique to Ocimum basilicum grown in Qatar.

Key words: Ocimum basilicum (Basil), Mashmoom, Natural products, Isolation and qualitative analysis.

INTRODUCTION

Since the dawn of civilizations, plant preparations have been used to treat diseases and various ailments. In this regard, various preparations of Ocimum plants are being used for the treatment of many ailments for more than 2000 years1Ocimum species belong to Lamiacea family.  Lamiacea is one of the largest families in the plant kingdom with ~220 genera and 3500 to 4000 species2Ocimum plants in this family belong to ~ 45 genera, represented by 574 species, and256 of them are endemic species3.Ocimum species, although native to tropics of Asia, are believed to have originated from India4, 5and as per recent reports they are grown and used all around the world given their versatile pharmacological properties. The pharmacological activities exhibited by various preparations of Ocimum species include antibacterial, antioxidant, antiulcer, antimalarial, antidiabetic, anti-inflammatory, anti-lipidemic, anticancer and immunomodulatory in nature6-8.  Further, some of the Ocimum plant species are also used as food flavoring agentsas well as fragrances in perfumery9, 10. Many plants grown in different geographic regions and varied conditions selectively produce phytochemicals, and this has been well documented in Ocimum species4, 5.  There are very few investigations involving Ocimum species leading to identification of natural products in plant species native to and/or adopted to grow in desert environment, and this is especially true with plants used in traditional medicine and home remedies in the Middle East region. The pharmacological/commercial value of the Ocimum species depends on the species, the geographical regions where it is grown, type and percent composition/amount of the amount of the bioactive phytochemicals present in the plant.  There are a very limited number of studies that have evaluated the chemical constituents of Ocimum species that have been adopted to grow in the desert environment of the Gulf region and GCC countries and these studies have mainly focused on the identification of essential oils and phenolic compounds11, 12.  Accordingly, the purpose of this study was to qualitatively evaluate the complete composition of phytochemical compounds present on the Ocimum bacilicum (locally known as Mashmoom) grown in Qatar.  In this regard we have analyzed methanolic and aqueous extracts of Ocimum basilicum for the presence of alkaloids, flavonoids, phenols, glycosides, tannins, terpenoids, saponins, anthraquinones, and phlobatatannins.

MATERIALS AND METHODS

Preparation of plant extracts

The aerial parts of the pant with leaves and flowers of the Ocimum basilicum were collected from a local garden, in Doha, Qatar.  The collected plant parts (leaves and flowers) were washed with fresh water and dried under shade at room temperature.  The dry plant was cut into small pieces and powered using a domestic varying dry grinder.  The powder obtained was stored in sterile containers at room temperature in a dehumidified chamber.

Preparation of methanol extracts

Methanolic extracts of the Ocimum basilicum was prepared using the Soxhelt extraction method.  Briefly, 50g of the powder preparation (above) was rolled into the Soxhelt cylinder using Whatman filter paper and compounds soluble in methanol were extracted by soaking the powder with 250 mL methanol for 24 h in a Soxhelt extractor.  The methanol was removed using a rotary evaporator.  The crude extracts thus obtained were dried on hot plate at 30oC to remove traces of solvent and stored at 4oC for the further analysis.

Preparation of Aqueous extracts

The aqueous extracts of the Ocimum basilicum was prepared by first suspending 25g of plant powder in 250 mL of distilled water followed by stirring the aqueous suspension at 30-40 oC for 30 min on water bath. At end of this process, the suspension was filtered using Whatman No. 1 filter paper, the filtrate was then centrifuged at 500 g for 15 min and the supernatant was stored in sterile bottles at 4oC.

Qualitative phytochemical analysis

The qualitative tests used herein were adopted from Sasidharan and associates13, and Kokate and associates14.

The methanolic and aqueous extracts, prepared as above, were tested for the presence of various bioactive compounds as follows.

Wagner’s reagent test for alkaloids

In this test, a small amount of the methanolic extract (1mg) dry powder residue was first dissolved in 2 mL of 2M HCl, the mixture was filtered and the filtrate was treated with Wagner’s reagent (iodine in potassium iodide). The formation of brown/reddish-brown precipitate is indicative of the presence of alkaloids.

In the case of aqueous extracts, 3 mL of filtrated aqueous extract was mixed with 3-6 drops of Wagner’s reagent. The formation of brown/reddish-brown precipitate is indicative of the presence of alkaloids.

Keller-Kilani testfor glycosides

In this test, small amounts of the crude extracts (2 mL) were mixed with 2 mL of glacial acetic acid containing 1-2 drops of 5% solution of FeCl3.  The mixture was then poured into test tube containing 1 mL of concentrated H2SO4.  Formation of a brown ring at the junction of the two liquid layers is indicative of the presence of glycosides.

Test for tannins

In this test, small amounts of the crude extracts (2 mL) were mixed with 2-3 drops of 5% of FeCl3 and formation of a greenish colored precipitate is indicative the presence of tannins.

Test for flavonoids

In this test, small amounts of the crude extracts (1 mL) were mixed with a few drops of 10% of NaOH solution. The formation of an intense yellow color is indicative of the presence of flavonoids.

Salkowski test for terpenoids/steroids

In this test, small amounts of the crude extracts (2 mL) were mixed with 2 mL of chloroform follow by a 2-3 drops H2SO4 a reddish brown ring at the junction indicate the presence of steroids.

Foam test for saponins

In this test, small amounts of the crude extracts (2 mL) were mixed with 5 mL of distilled water in a test tube and shaken vigorously and allowed to stand for 10 min on a counter top.  Presence of saponins will produce froth.

Test for phenols

In this test, small amounts of the crude extracts (2 mL) were mixed with 2mL of the 5% FeCl3to determine the presence of phenols.  Formation of a blue-green or black color indicates the presence of phenols in the samples.

Test for anthraquinones

In this test, small amounts of the crude extracts (2 mL) were mixed with a small quantity (1 mL) of benzene and 3-5 drops of 26% NH3. Formation of a pink or violet or red coloration in the ammonical layer is an indicator of the presence of anthraquinones.

Test for anthocyanins

In this test, small amounts of the crude extracts (2 mL) were mixed with 2 mL HCl and ammonia (NH3).  Formation of a pinkish red to bluish violet coloration is indicative for the presence of anthrocyanins.

Test for phlobatatannins (precipitate test)

In this test, small amounts of the crude extracts (2 mL) were mixed with 2 mL of 1% of the HCl in a test tube and the tube was placed on a boiling water bath. Formation of a red precipitate is indicative the presence of phlobatatannins.

Xanthoproteic test for proteins

In this test, small amounts of the crude extracts (2 mL) were mixed with 1mL of concentrated H2SO4. Formation of a white precipitate is indicative of the presence of protein.

RESULTS AND DISCUSSION

Qualitative analysis (eleven tests listed in Table 1) for presence of important phytochemicals in the aqueous and methanolic extracts of Ocimum basilicum grown in Qatarindicates the presence of a variety of compounds (Table 1).  Glycosides, tannins, terpenoids, phenols and phlobatatannins were present in both methanolic and aqueous extracts of Ocimum basilicum grown in Qatar.  Alkaloids, flavonoids, saponins, and anthraquinones were only found in methanolic extracts but not in aqueous extracts.  Anthrocyanins and proteins were absent in both methanolic as well as aqueous extracts. An interesting observation is that the anthraquinones test resulted in the formation of greenish-yellow precipitate formation when methanolic extracts were tested for anthraquinones rather than pink- violet or red coloration. This suggests that the methanolic extracts may contain new or modified anthraquinones compounds; accordingly we believe that the Ocimum basilicum grown in Qatar is likely to contain unique anthraquinones.

Table 1: Phytochemical analysis of Ocimum basilicum grown in Qatar

Item No. Test Procedure Observation Methanolic extract Aqueous extract
1 Alkaloids Wagner’s reagent Brown/reddish ppt. +ve -ve
2 Glycosides Keller-Kilani reagent Brow ring at the junction +ve +ve
3 Tannins Braymer’s test Greenish ppt. +ve +ve
4 Flavonoids Drop of 10% NaOH solution Intense yellow color +ve -ve
5 Terpenoids Salkowski test Reddish brown ring at the junction +ve +ve
6 Saponins Foam test Stable froth produced +ve -ve
7 Phenol 2mL 5% FeCl3 Blue-green +ve +ve
8 Anthraquinones Benzene + 26% NH3 Greenish yellow precipitate* +ve -ve
9 Phlobatatannins 2mL 1% HCl + heat Red ppt. +ve +ve
10 Anthocyanins 2mL HCl (2M) + ammonia (NH3) No change -ve -ve
11 Proteins Xanthoproteic test No change -ve -ve

*the anthraquinones are expected to produce a pink- violet or red coloration in ammonical layer, but greenish-yellow precipitate formation was observed.

Previous studies reported that flavonoids, saponins, tannins, terpenoids, and alkaloids are the most phytochemical compounds present in wild type Ocimum species. These phytochemicals, e.g., alkaloids8, tannins9 and saponins10, have remarkable cancer chemopreventive and anticancer15-17 activities.  The phenols and flavones have been shown to have antioxidant18-20 and anti-inflammatory21, 22 activities.  Flavonoids have been used to treat the stress related ailments and as dressings for wounds normally encountered in circumcision rites, bruises, cuts and sores23, 24.  Steroids are very important compounds due to their relationship with reproductive hormones25, 26.  Given the above medicinal uses of important phytochemicals, and the fact that these compounds are present in the Ocimum basilicum grown in Qatar, the plant extracts are likely to have high anti-oxidative, anti-microbial, anti-inflammatory, anti-mutagenic, anti-viral, and anti-allergic activities. This preliminary screening of the phytochemical compounds of the Ocimum basilicum grown in Qatar will serve as basis for further work leading to quantitative analysis and identification of individual compounds.  This will involve testing of the extracts and various fractions for antioxidant and anticancer activity. Further work will involve isolation, purification, and characterization of active constituents responsible for the various activities exhibited by this plant extracts.

Conclusions

The qualitative analysis aqueous and methanolic extracts of Ocimum basilicum grown in Qatar has revealed the presences of glycosides, tannins, terpenoids, phenols, and phlobatatannins in both extracts.  In addition, the methanolic extracts also contained alkaloids, flavonoids, saponins and anthraquinones.  On the other hand, neither extracts contained proteins or anthrocyanins.  The anthraquinones present in Ocimum basilicum appear to be are unique.  Further work leading to quantitative analysis of various phytochemical listed above and their identity is being undertaken.

Acknowledgements

The authors thankfully acknowledge the grant support from Qatar University (QUUG-CAS-DCES-15\16-11) for this work.

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