**9.5 Glycosides**

Glycosides are relatively polar, and their polarity depends on both the number and type of sugar moieties attached to the aglycone. Cardiac glycosides have bulky steroidal aglycone, which are soluble in chloroform. However, most glycosides are extracted using polar solvents like acetone, methanol, ethanol, water or mixtures of these solvents. When extraction in done using water as solvent, enzymatic breakdown can happen. This will be avoid by using boiling water or add important proportions of alcohol or ammonium sulfate to the extract. In some cases, it may be the hydrolytic separation of the aglycone and sugar before or after extraction [122, 123].

#### **9.6 Total phenolic and total flavonoids content**

Phenolic compounds are well-known phytochemicals found in almost all plants. They can be simple phenols, benzoic and cinnamic acid derivatives, coumarins, tannins, lignins, lignans, and flavonoids [124]. Flavonoids are a group of plant constituents, the most common phenolic compound produce by plants as secondary metabolites in response to diverse biotic and abiotic factors [63, 82, 124]. They are responsible for the characteristics of flavor, color and pharmacological activities [67, 80, 125]. Because of their positive effects on human and animal health, and medical application for disease therapy and chemoprevention, interest in flavonoids increases [126, 127]. Complete extraction of phenolics is the next critical step after the sample preparation. The most common procedures of extraction of phenolics employ solvents, either organic or inorganic. Different parameters may influence the extraction yield, that includes temperature, the solvent used, time, solvent-to-sample ratio, as well as the number of repeated extractions of the plant material [124].

There is no universal extraction method and each optimized procedure is unique [82]. Due to the complex nature of the sample matrix and diverse chemical characteristics of flavonoids, it is consensual among scholars that there is no single or/and standard method to be used for every material or flavonoids to be extracted at present [67]. Maceration, water infusion, and Soxhlet extractions are generally used in research laboratories and/or in small manufacturing companies. The choice of solvent for extraction such as water, acetone, ethyl acetate, alcohols (methanol, ethanol, and propanol), and their mixtures will influence phenolics' extraction [124, 128]. The extraction of flavonoids-containing sample material are still performed by simple direct solvent extraction. It can also be extracted in a Soxhlet apparatus, first with *n*-hexane or diethyl ether to remove fats, and then with ethyl acetate or ethanol to obtain total phenols. This procedure is unsuitable for thermolabile components. A commodious and frequently used technique is sequential solvent extraction. Dichloromethane is used in the first step to remove flavonoid aglycones and non-polar components. A subsequent step using alcohol or alcohol-water mixtures will therefore extract flavonoid glycosides and other polar constituents. Cowan indicated that acetone was the most selective solvent for extracting flavonoids [129]. Anokwuru and collaborators discovered that acetone and *N*,*N*-dimethylformamide (DMF) were highly influential for removing antioxidants [130]. In most cases, flavonoids and polyphenols are coextracted [82]. Furthermore, several promising methods (Microwave-assisted extraction (MAE), Enzyme-assisted extraction (EAE), Pressurized liquid extraction (PLE), Ultrasound-assisted extraction (UAE), Matrix solid-phase dispersion (MSPD), and Supercritical fluid extraction (SFE) are nowadays used with increased yields and lower cost as main advantages [8, 82].

Due to the multiplicity of hydroxyl functions, phenols tend to be relatively polar and dissolve in aqueous alcohols. They may also be extracted or partitioned into aqueous alkali as phenolate salts as they are weak acids. A problem encountered with phenolic compounds is that they can undergo extensive polymerization reaction by polyphenol oxidation. This reaction is responsible for developing brown coloration in damaged plant material when exposed to the air and in certain extracts. The polymerization reaction is catalyzed by acid [131].

#### **9.7 Total mixture of crude saponins**

The procedure for isolating mixtures of crude saponins (i.e., steroidal or triterpene glycosides) is shown in **Figure 15**. Fats are removed from the plant material by treating with *n*-hexane and after extraction with methanol. The resultant methanol extract

*Extraction of Bioactive Compounds from Medicinal Plants and Herbs DOI: http://dx.doi.org/10.5772/intechopen.98602*

#### **Figure 15.**

*General fractionation procedure to obtain a precipitate of crude saponin from plants, adapted from the literature [132].*

is evaporated under vacuum and suspended in deionized water (presaturated with n-butanol), and partitioned with *n*-butanol. Diethyl ether is added to the butanol solution to precipitate the saponins [3, 132]. Selective extraction and fractionation of plant sterols (including sapogenins, bufadienolides, and cardiac glycosides) using manipulations and liquid/liquid partitioning have been described [3, 133]. Partitioning between the aqueous phthalic anhydride and organic solvent can be used to separate alcohols from non-alcohols. The alcohols partition into the aqueous layer as half-phthalates and can be regenerated by treatment with sodium methoxide in methanol. Sterols with ketone functional groups can be set-apart from non-ketones by liquid/liquid partition between organic and aqueous layers using Girard's hydrazide reagents (H2N.NH.CO.CH2.NR3 + Cl− ), and generate ketones by acid hydrolysis [3, 133].

## **10. Conclusion**

There is a clear and growing interest in the extraction procedure of natural products and their isolation, identification, and applications. Research innovation and

safe extraction processes are of primary importance in modern analytical processes, which are economically viable and environmental friendly. In the process of plant extracting plant material, it is peremptory to reduce interference of components that may be co-extracted with the target compounds, and to bypass contamination of the extract, moreover to prevent degradation of necessary metabolites or the formation of artifact as a result of extraction conditions or solvent impurities. Regardless of the extraction procedure, the resulting solution should be filtered to remove any particulate matter. Plant extracts should be stored for short time at room temperature or in sunlight to avoid increasing risks associated with the production of artifact making and additionally degradation or isomerization of extract components. The most suitable extraction procedure depends on the matrix of the plants and the type of compost, and should follow clear selection criteria.
