**5. Quantification of phytochemicals of essential oils of** *Cymbopogon* **species**

Refs. [60, 61] have documented the quantitative determination of phytochemicals (total alkaloids, flavonoids, phenol, saponins and tannins) of extracts of leaves using spectrophotometric and Folin-Ciocalteu methods, expressed per gram of the sample dry matter [60, 61]. These were majorly initiated by ethanolic and methanolic of acidified methanolic extractions as necessitated by the phytochemical of interest [62], followed by centrifugation and storage at −20°C until analysis was done.

For instance, the 1,10-phenanthroline method of total alkaloids content (TAC) estimation, as described by Ref. [63], entails the oxidation of alkaloids by iron (III) and subsequent complexation of iron (II) with 1,10-phenanthroline, to form a red-colored complex having the maximum absorbance at 510 nm. The reaction mixture containing 1 ml plant extract, 1 ml of 0.025 M FeCl3 in 0.5 M HCl, and 1 mL of 0.05 M of 1, 10-phenanthroline in ethanol is usually incubated for 30 min in hot water bath with maintained temperature of 70 ± 2°C, before the measurement of the absorbance of red-colored complex at 510 nm against reagent blank. Alkaloid contents are then estimated and expressed as the standard curve of quinine (0.1 mg/ ml, 10 mg dissolved in 10 ml ethanol, and diluted to 100 ml with distilled water) and the values expressed as g.100 g–1 of dry weight. This simple, sensitive, and economically viable spectrophotometric method has been used in the determination of some *Rauwolfia* alkaloids (ajmaline, ajmalicine, reserpine, and yohimbine HCl) in tablets of pharmaceutical formulations, with reports showing that the common excipients do not interfere with the proposed method. A statistical comparison of these results with the results of the reported approach shows good agreement and no significant difference in accuracy [61–63].

Phenolic compounds inhibit lipid oxidation by scavenging free radicals, chelating metals, activating antioxidant enzymes, reducing tocopherol radicals, and inhibiting enzymes that cause oxidation reactions. These may provide the basis for the rapidly growing interest in the use of natural antioxidants and antimicrobials [64]. Quantification of total flavonoids content (TFC) has been according to the aluminum chloride method reported by [65], involving the dispense of 0.5 ml of extract into test tube, followed by the addition of 1.5 ml of methanol, 0.1 mL of aluminum chloride (10%), 0.1 ml of 1 M potassium acetate, and 2.8 ml of distilled water in a reaction

mixture. The absorbance read at 514 nm, after allowing to stand at room temperature for 30 min, is expressed as quercetin equivalent (QE) in mg/g material. In the same vein, the total phenolic content (TPC) quantification of samples extracts can be determined according to the Folin-Ciocalteu method of [62], where 1.5 ml of a 1 in 10 dilution of Folin-Ciocalteu reagent is added to 300 ml of leaf sample extract, followed by 1.2 ml of Na2CO3 solution (7.5 w/v). The absorbance read, at 765 nm against a blank after allowing to stand at room temperature for 30 min, is expressed as gallic acid equivalent (GAE) in mg/g material.

Variations in the extraction yields could arise from the different extraction methods and solvents. Other factors could be the evaluated variety, harvest year, processing, and storage [66]. Using walnut leaf as a case study, Ref. [67] investigated several solvents with different polarities such as hexane, chloroform, ethyl acetate, methanol, and ethanol for the evaluation of the cytotoxicity of walnut leaf extract on human cancer cell lines. They reported that the resulting methanol extract had the highest amount of TPC and TFC (120.28 ± 2.32 and 59.44 ± 0.87 mg/g DE, respectively) using colorimetric methods. For the ethanolic extracts, the concentration of the phenolic compounds in young leaves was substantially greater than those in the mature leaves. Employing the response surface methodology of optimization of the ultrasound assisted hydroalcoholic extraction of phenolic compounds of walnut leaves, Ref. [68] tried to establish the optimum conditions and the maximum predicted TPC, using 61% ethanol concentration, 51.28 min extraction time, and the 4.96 v/w liquid-tosolid ratio to obtain 10,125.4 mg GAEs/l, while 2925 mg quercetin equivalents (QEs)/l as maximum TFC was achieved by using ethanol with 67.83% concentration, v/w liquid-to-solid ratio of 4.96, and 49.37 min of extraction time. Under these conditions, the experimental results were reasonably similar to the values predicted by the polynomial response surface model equation. Ref. [69] compared the antioxidant and antimicrobial activities of the prepared ethanol and water extracts from the leaves of three plants, namely *P. aphylla*, Persian walnut, and oleander. They showed that the ethanol extracts had the highest amount of total phenolics and flavonoids in all assays, as well as highest antioxidant and antimicrobial activities when compared with the water extracts.

Initially, the formation of stable and persistent foam on the liquid surface for approximately 15 min represented the presence of saponins [70]. However, the quantification of plant saponins is usually performed by spectrophotometry and chromatography. The major difference in quantitative expression between the two techniques is that spectrophotometry quantifies the total saponin value, while chromatography quantifies specific saponin compound [71].

#### **5.1 Spectrophotometric method of phytochemical quantification**

Regarded as a simple, rapid, and inexpensive approach, total saponins assay, also known as vanillin-sulfuric acid assay, involving the reaction of oxidized triterpene saponins with vanillin is one of the spectrophotometric methods used to quantify saponins. It uses sulfuric or perchloric acid as oxidant, resulting in a distinctive purple coloration of the reaction system [71]. Being the most commonly utilized spectrophotometric method for quantifying plant saponins and providing an excellent reference for future experimental design, reports suggest that in order to allow full color development, few criteria, such as choice of standards and wavelength, should be taken into account when selecting this method [70]. However, researchers have reported 544-nm wavelength, with a majority selecting wavelengths in the range *Phytochemical Contents of Essential Oils from* Cymbopogon *Species: A Tropical Medicinal Plant DOI: http://dx.doi.org/10.5772/intechopen.105396*

of 480–610 nm (excluding 473 nm and 283 nm). This is likely due to the maximum absorption of purple color that falls within this range [72, 73].

Hemolytic method is a spectrophotometric method for determining the saponin concentration of a plant material [74]. This is based on the release of oxy-hemoglobin when saponins react with blood reagent producing measurable color changes detected by the spectrophotometer. Ref. [75] have quantified the saponin content in bitter gourd varieties with the hemolytic technique, with their result showing that white bitter gourd types had much lower levels of saponin (0.25%) than the green varieties (0.67%). In furtherance, the saponin extract was dissolved in distilled water, before incubation of 100 μl of this solution with 1 ml fresh EDTA-blood at 30°C for 30 min. Hemoglobin was measured in the supernatant photometrically at 545 nm, after centrifugation for 10 min, and the result is expressed in hemolytic saponins.

#### **5.2 Chromatographic method of phytochemical quantification**

Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and ultra-pressure liquid chromatography (UHPLC) are the most common chromatographic methods employed [73, 76, 77]. Multiple reaction monitoring (MRM)-based UHPLC-ESI-MS/MS technology, according to studies, was developed to provide more precise and sensitive measurement of main saponins and sapogenins in conjunction with chlorogenic acid [78]. Refs. [79, 80] described the use of ultra-high-performance liquid chromatography coupled to single-stage Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap-MS) to simultaneously detect and quantify phytochemicals in green tea and walnut leaves-derived nutraceuticals. Ref. [81] submit that by combining LC separation and MS detection, a high selectivity could be attained since the MS detector's selectivity allows for more precise identity by confirmation by comparing fragmentation patterns and observing qualifier and quantifier ion transitions.

Additionally, although sensitivity—the connection between analyte signal and concentration—may not be a crucial parameter for evaluation during method validation, it provides information about the instrument signal and might be helpful during method optimization. In light of these, Ref. [78] suggest that this method can be used to quantitatively measure bioactive compounds in crude plant materials and other related products, while also determining the same compounds in other biological sample matrices such as plasma, potentially minimizing matrix effect. Reports of Refs. [82, 83] show chromatographic methods to allow separation and purification of various saponin biotypes from plant materials to identify a specific saponin compound and investigate its pharmaceutical property. Refs. [84, 85] suggest that the main goal of all the studies using HPLC technique is the quantification of specific saponin components. The specific saponin content detected serves as an excellent data reference source to future researchers, in addition to providing a reliable scientific reference to pharmaceutical manufacturers interested in further processing of their respective plant sources [71].

Standardization and purification of complex extracts are still problematic since the mixtures are more toxic than individual components and present more difficulty in detoxification than a single molecule [86, 87]. More so, isolation, synthesis, or formulation processes could be slow and expensive, but relatively inexpensive for plant essential oils. In light of these, Ref. [88] allude that simultaneous quantification and qualitative analyses of phytochemicals could easily be achieved *via* quick and conventional methods such as non-destructive near-infrared spectroscopy and

isocratic high-performance liquid chromatography. These improved methods can support rapid and precise content evaluation and confirmation [86].
