Qualitative Analysis of Phytochemicals from Sea Buckthorn and Gooseberry Qualitative Analysis of Phytochemicals from Sea Buckthorn and Gooseberry

This chapter describes in detail recent research results obtained from the qualitative screening of different phytochemicals found in aqueous extracts of sea buckthorn and gooseberry, fruits with important pharmacological effects due to their high content in vitamin C. Phytochemical investigations reveal the presence of active principles (e.g., saponins, flavonoids, alkaloids, carbohydrates, terpenoids, etc.) in sea buckthorn and gooseberry and are accomplished by using well-established standard methods. All these qualitative determinations rely on the visual color change reaction as a basic response to the presence of a specific phytochemical compound. The active principles from sea buckthorn and gooseberry are extracted according to a well-settled extraction method, which involves infusing the fruits in an aqueous medium, for 24 h, at a constant temperature of 4°C. few drops of concentrated sulfuric acid are slowly dripped and the resulted solution is shaken carefully. The appearance of a violet ring at the interface of the two liquids indicates the presence of carbohydrates in the aqueous extracts. In the case of sea buckthorn aqueous extract, the solution turns purple-red and a brown precipitate is obtained from gooseberry aqueous extract.


Introduction
Phytochemistry, basically described as the chemistry of plants and different plant parts, is generally considered an early subdivision of organic chemistry and is very important in the identification of plant compounds with medicinal properties [1].
Phytochemistry is associated with numerous species of secondary metabolites produced in plants by biosynthesis and the natural combination of all these secondary metabolites gives the general beneficial therapeutic effects of that specific plant [2,3].
Plants biosynthesize phytochemicals to protect themselves from insect attacks and plant diseases. Phytochemicals ("Phyto" is the Greek word for plant) are plant chemicals with no nutritional value, non-essential nutrients, and with disease preventive properties. Some of the most common phytochemicals are lycopene (found in tomatoes), flavonoids (found in fruits), and isoflavones (found in soy) [4,5].
Species belonging even to the same genus can differ one from another in different proportions and sometimes these differences are subtle and extremely difficult to determine. Therefore, new phytochemical methods quickly developed coming in addition to those that were already known and applied [6,7].
There are many known phytochemicals, and each has its own possible action [8][9][10]: • antioxidant: protect human cells from oxidative stress thus considerably reducing the risk of developing numerous types of cancer; • hormonal action: isoflavones are able to imitate human estrogens, reducing the symptoms of osteoporosis; • antibacterial: can be used as alternative therapy against infections caused by different bacteria; • physical action: many phytochemicals physically attach to cell walls thus preventing the adhesion of pathogens.
Sea buckthorn (Hippophae rhamnoides L.), an ancient plant with modern attributes, has numerous pharmacological effects: cardioprotective, inhibits platelet aggregation, lowers the levels of cholesterol and blood pressure, and provides antioxidant activity. The berries have an orangeyellowish color (see Figure 1a) and are an important source of vitamin C and A, phenolic compounds (especially flavonoids), and phytosterols [11,12]. The mineral content (whether it's the fruit itself or the juice) is another important factor, which comes to complete all the beneficial properties of sea buckthorn: five essential minerals (calcium, iron, magnesium, sodium, and manganese) and four trace elements (chromium, vanadium, selenium, and cobalt) [13].
Although both sea buckthorn and gooseberry are used in traditional medicine for the treatment of various diseases, no clear scientific evidence exists to prove their therapeutic benefits and, therefore, it is very important to determine the qualitative content of these two fruits.
In this chapter, sea buckthorn and gooseberry dried fruits are used to prepare aqueous extracts using a method that involves the cold infusion at a constant temperature of 4°C for 24 h. The two aqueous extracts are further used for the qualitative screening of phytochemicals, and the most important bioactive chemical constituents that are studied are carbohydrates, flavonoids, alkaloids, glycosides, steroids, tannins, proteins, amino acids, and terpenoids. All these qualitative studies use standard analytical methods and the results are clearly detailed in the present chapter.

Preparation of aqueous extracts from sea buckthorn and gooseberry
Sea buckthorn (Hippophae rhamnoides L.) and gooseberry (Ribes grossularia) are bought readily dried from local natural shops and are further used to prepare aqueous extracts using a method that involves the following steps (Figure 2): grinding the dried fruits into a fine powder, weighting an exact amount of powder, and extracting it using a determined volume of distilled water at a constant temperature of 4°C.
The cold infusion takes place in sealed "French press" type coffee filters (Figure 3), one for every fruit involved in this research [17].  The two extracts were left to incubate for 24 h so that as much of sea buckthorn and gooseberry as possible could be transferred to the aqueous extracts. The aqueous extracts thus prepared were separated, filtered, and the volumes of the resulted aqueous extracts were measured. An additional vacuum filtration was carried out so that all debris were removed from the aqueous extracts.
The sea buckthorn aqueous extract and the gooseberry extract were kept in the refrigerator for more than 12 weeks for further use, without any alteration.
The extractive value (yield percentage) of the sea buckthorn and gooseberry samples were weighted before and after the preparation of the aqueous extracts and the results are presented in  The pH was measured for the two aqueous extracts and the value was 6.5 for sea buckthorn as well as for gooseberry aqueous extracts. where W 1 = net powder weight (g) resulted after the aqueous extraction and W 2 = total powder weight (g) used for the preparation of sea buckthorn and gooseberry aqueous extracts.
The volume of the resulted aqueous extracts was measured (mL) and compared to the initial volume of distilled water ( Table 2).

Qualitative screening of phytochemicals from sea buckthorn and gooseberry
Different qualitative phytochemical analyses are known that allow, by using standard analytical techniques, the determination of chemical groups, or compounds in aqueous extracts from different plants. These qualitative tests are based on color or precipitation reactions as a positive response to the presence of those specific chemical compounds [19,20]. All the color reactions allow only determining the presence or absence of various chemical groups and not the amount in which they are present in different aqueous extracts.
Standard qualitative methods are used to analyze qualitatively the aqueous extract prepared from sea buckthorn and gooseberry [21,22]. Carbohydrates are usually neutral, water-soluble chemical compounds, but there are some exceptions and some, such as pectic acid, gluconic acid, or alginic acid, are acidic in the living world.

Qualitative screening of carbohydrates
There are different standard phytochemical methods used for the qualitative screening of carbohydrates found in aqueous extracts [24]. The results obtained for sea buckthorn and gooseberry aqueous extracts are fully described in Table 3.

General screening of carbohydrates
Experimental: 1 ml Molisch reagent (a solution of α-naphthol in ethanol) is added to 2 ml aqueous extract and few drops of concentrated sulfuric acid are slowly dripped and the resulted solution is shaken carefully. The appearance of a violet ring at the interface of the two liquids indicates the presence of carbohydrates in the aqueous extracts.
In the case of sea buckthorn aqueous extract, the solution turns purple-red and a brown precipitate is obtained from gooseberry aqueous extract.

Detection of reducing sugars
The general definition of reducing sugars is any type of sugar that can act as a reducing agent due to the free aldehyde or ketone groups. All monosaccharides are reducing sugars, along with some di-, oil-and polysaccharides. Several tests are available for detecting reducing sugars in aqueous extracts (Figures 4 and 5) ( Table 3) [25].

Phytochemical test Sea buckthorn Gooseberry
Carbohydrates (     g. Moore test: this test particularly reveals the presence of glucose. To 2 ml of aqueous extract an equal volume of 5% NaOH is added and the mixture is boiled for 5 min with. The solution has initially a yellow coloration that changer to reddish-brown.
By performing Molisch's test, it reveals that both aqueous extracts contain different classes of carbohydrates. Specific qualitative test for carbohydrates reveals the presence of monosaccharides in gooseberry aqueous extract and of di-, oil-and polysaccharides in both sea buckthorn and gooseberry extracts.

Detection of hexose sugars
Hexoses are monosaccharides that contain six carbon atoms and are divided into aldohexoses and ketohexoses depending on the functional group [26]. Three qualitative methods reveal the presence of hexose sugars and the results are presented in Table 4.
a. Seliwanoff test: to 1 ml of aqueous extract, 3 ml of Seliwanoff's reagent (a mixture of resorcinol in hydrochloric acid) is added and boiled for 2 min. A red solution is obtained indicated a positive reaction (Figure 6) As it is clear from the Table 4, hexose sugars are present in both sea buckthorn aqueous extract as well as in gooseberry aqueous extract.

Qualitative screening of tannins and phlobatannins
Most of the tannins, a group of phenol compounds usually found in plants, are soluble in water. Phlobatannins are considered a novel class of ring-isomerized condensed tannins [17].
The test for tannins is generally described as [27]: to 1 ml aqueous extract 2 ml of 5% ferric chloride are added and a dark-blue or greenish-black color appears.
Phlobatannins are tested following a standardized method: to 1 ml aqueous extract of sea buckthorn and gooseberry few drops of diluted HCl (1%) is added and a red precipitate should appear ( Table 5).
Tannins are present in both aqueous extracts, while small traces of phlobatannins can be found in gooseberry aqueous extract.

Qualitative screening of saponins
The general method involved in the qualitative analyze of saponins is: 2 ml of aqueous extract and 2 ml of distilled water are shaken for 15 min in a graduated cylinder. A 1 cm foam layer is a positive response to the presence of saponins (see Table 6).
Qualitative screening of saponins in aqueous extracts from sea buckthorn and gooseberry revealed that only the second one contains saponins.

Qualitative screening of flavonoids and phenolic flavonoids
Flavonoids have important functions in plants: attract pollinating insects, fight against different microbial infections, and control cell growth [28].
Flavonoids are tested according to the following method: 2 ml aqueous extract and 1 ml of 2N sodium hydroxide are mixed. A yellow color indicates the presence of flavonoids.

Phytochemical test Sea buckthorn Gooseberry
Carbohydrates (hexose sugars)-Seliwanoff The test for phenolic flavonoids (Figure 7): 1 ml aqueous extract is mixed with 2 ml of 10% lead acetate solution and a brown precipitate indicates a positive response (see Table 7).
Flavonoids are present in both aqueous extracts (sea buckthorn and gooseberry), while phenolic flavonoids are present as small traces in gooseberry aqueous extract.

Qualitative screening of alkaloids
Alkaloids are a group of basic plant bioactive compounds that possess an N-containing heterocycle, are generally colorless, crystalline, insoluble in water but soluble in many organic solvents [29].

Phytochemical test Sea buckthorn Gooseberry
Tannins

Phlobatannins
Pale pink solution Red-orange solution Table 5. Qualitative screening of tannins and phlobatannins.

Phytochemical test Sea buckthorn Gooseberry
Saponins 0.2 cm foam layer 1.5 cm foam layer Table 6. Qualitative screening of saponins.  b. Mayer test: to 1 ml aqueous extract, 2 ml concentrated HCl is added followed by few drops of Mayer's reagent (a solution of mercuric chloride and potassium iodide in water); a green color or white precipitate indicates the presence of alkaloids (the results are presented in Table 8).
c. Hager test: 2 ml aqueous extract and 2 ml Hager's reagent (a saturated aqueous solution of picric acid) are mixed together and a yellow precipitate indicates a positive test.
According to the results presented in Table 8, alkaloids are absent from all the aqueous extracts.

Qualitative screening of anthraquinones and anthocyanosides
The method used for the qualitative screening of anthraquinone compounds involves the reaction of 1 ml aqueous extract with a few drops of 10% ammonia solution with the formation of a pink precipitate.
Anthocyanosides are present when a pink color appears after the reaction between 1 ml aqueous extract with 5 ml dilute hydrochloric acid (1%). The results are detailed in Table 9.

Phytochemical test Sea buckthorn Gooseberry
Alkaloids-Wagner Red-brown solution Red-brown solution

Alkaloids-Mayer Light-yellow solution Red-brown solution
Alkaloids-Hager Clear yellow solution Red-brown solution Table 8. Qualitative screening of alkaloids.

Phytochemical test Sea buckthorn Gooseberry
Anthraquinones Green-yellow solution Brown-yellow solution

Anthocyanosides
Light yellow solution Brown-yellow solution Table 9. Qualitative screening of anthraquinones and anthocyanosides.

Flavonoids Green-yellow solution Light brown yellow solution
Phenolic flavonoids Light yellow solution Opalescent brown-yellow solution Table 7. Qualitative screening of flavonoids and phenolic flavonoids.
According to the results presented in Table 9, anthraquinones and anthocyanosides are absent from both aqueous extracts.

Qualitative screening of proteins and amino acids
Proteins are involved in all physiological processes that take place in all living cells. Proteins are colloidal, do not diffuse through the plasma membrane, are irreversible coagulated upon heating and are insoluble in neutral salts [30].
Amino acids are amphoteric phytocompounds, highly reactive, with an amino and carboxylic acid moiety, therefore, being mostly water soluble.

General screening of proteins and amino acids
Experimental: 1 ml aqueous extract reacts with 5-6 drops of Millon's reagent (mixture of mercuric nitrate, mercurous nitrate, concentrated nitric acid, and distilled water) and a white precipitate is formed that changes its color to red upon heating. Millon's test is a non-specific test for detecting proteins and amino acids (tyrosine) and, therefore, it must be confirmed by other qualitative tests.
The results obtained after the two aqueous extracts react with Millon reagent are as follows: an opalescent orange solution in the case of Sea buckthorn and a red-brownish precipitate in the case of Gooseberry, therefore confirming the presence of small amounts of proteins and/ or aminoacids in Gooseberry aqueous extract.

Detection of amino acids
There are two different standard methods used (see results in Table 10): a. Ninhydrin test: take 3 ml aqueous extract and mix it with three drops of 5% lead acetate solution then heat the resulted solution. A purple or blue coloration indicates a positive reaction (Figure 8).
b. Test for cysteine: 5 ml aqueous extract is boiled with a small amount of 40% NaOH and few drops of 5% lead acetate solution are added. A black precipitate is formed.
The test for cysteine gives a positive reaction in the case of sea buckthorn, while ninhydrin test is negative for both aqueous extracts.

Phytochemical test Sea buckthorn Gooseberry
Proteins and amino acids-

Detection of proteins
There are two different standard methods used (see results in Table 11): a. Biuret test: to 3 ml aqueous extract, 3 ml 4% sodium hydroxide solution, and few drops of 1% copper sulfate are added to form a purple solution.
b. Xanthoproteic test: to 3 ml aqueous extract, 1 ml of concentrated H 2 SO 4 is slowly dropped. A white precipitate appears that turns yellow upon boiling and orange after 1 ml of NH 4 OH solution is added.

Qualitative screening of steroids and terpenoids
The general procedure to test the presence of steroids is: to 1 ml aqueous extract, 10 ml chloroform is added and then slowly 10 ml sulfuric acid is dripped. Upper layer turns red and sulfuric acid layer turns yellow-green.
Terpenoids are analyzed by reacting 1 ml aqueous extract with 2 ml of chloroform and then, slowly, few drops of concentrated sulfuric acid. An interface with a reddish-brown coloration appears ( Table 12). The change in color can be observed in Figure 9.
The qualitative screening of steroids revealed that these phytochemicals are absent from all the extracts while very small traces of terpenoids could be visually observed in gooseberry aqueous extract.

Phytochemical test Sea buckthorn Gooseberry
Proteins and amino acids-Millon Opalescent orange solution Red-brownish precipitate

Proteins-biuret test Green solution Brown solution
Proteins-xanthoproteic test Opalescent brown solution Dark-brown precipitate Table 11. Qualitative screening of proteins.

Qualitative screening of glycosides
There are three different standard phytochemical methods: a. FeCl 3 reagent: the test is for cardiac glycosides: 1 ml aqueous extract, 1 ml FeCl 3 reagent (1 ml 5% FeCl 3 solution mixed with 99 ml glacial acetic acid) and few drops of concentrated H 2 SO 4 gives a greenish-blue color that appears in time.
b. Keller-Killiani test: the test is for cardiac glycosides: 5 ml aqueous extract, 2 ml glacial acetic acid, a drop of FeCl 3 solution, and 1 ml concentrated H 2 SO 4 form a brown ring and often a purple ring appears below (see results in Table 13).
c. Borntrager test: this test reveals the presence of anthraquinonic glycosides: 2 ml aqueous extract react upon boiling with 2 ml H 2 SO 4 . The solution is filtered, and equal volumes of chloroform are added and shaken vigorously, and two layers can be clearly observed. The organic layer is separated, and ammonia is added to form a pinkish-red color as a sign of positive reaction.

Conclusions
This chapter describes the qualitative phytochemical screening of two aqueous extracts prepared from dried fruits of sea buckthorn and gooseberry, plants with the important pharmacological properties and rich in nutrients. The qualitative screening consists of standard methods that are able to determine whether a phytochemical is present or not in the aqueous extracts.
The two aqueous extracts are obtained after a cold infusion at a constant temperature of 4°C for 24 h and are kept at the refrigerator for more than 12 weeks without alteration.
The general screening of carbohydrates revealed that, in the case of sea buckthorn aqueous extract, the solution turns purple-red and a brown precipitate is obtained from gooseberry aqueous extract. Molisch's test revealed that both aqueous extracts contain different classes of carbohydrates. Specific qualitative test for carbohydrates reveal the presence of monosaccharides in gooseberry aqueous extract and of di-, oil-and polysaccharides in both sea buckthorn and gooseberry aqueous extracts.
Alkaloids are absent from both extracts, while cardiac glycosides are present. The test for cysteine gives a positive reaction in the case of sea buckthorn, while ninhydrin test is negative for both aqueous extracts.
The results obtained when aqueous extract from sea buckthorn reacts with Millon reagent is an opalescent orange solution and, in the case of gooseberry aqueous extracts, a red-brownish precipitate is formed, thus confirming that small amounts of proteins and/or amino acids are present in gooseberry.