INTRODUCTION:

Plants are able to synthesize many chemical compounds that are referred to "secondary metabolites”¹. These compounds play an crucial role in the development of several useful medicines².

Achillea is a group of flowering plants belong to the family of Asteraceae, known as yarrow. The genus was named after Achilles, a Greek mythological character. According to folk tales, yarrow was used by Achilles' soldiers to treat their wounds³, hence allheal and bloodwort are some of the common names used. Many of the Achillea plant species are medicinal which have curative applications⁴. Search in literature showed many reports on immunological pharmacological, therapeutic and biological activities of this valuable herb⁵.

Since the widespread of Achillea species all over the world it have been utilized by native inhabitants as folk or traditional herbal medicines^6,7.

The plant family is of economic importance, it is used for the production of many oil products, vegetables, ornamentals and as medicinal plants^8,9. The species of interest is Achillea santolina, an everlasting herb mostly found in dry environments of Iraq and Jordan, it may also grow in colder and more humid weather in the regions north of the equator, such as Europe and Asia. Achillea santolina is rich in flavonoids¹⁰. and sesquiterpene lactones¹¹. The species also produces alkaloids, saponins, resins, tannins, sterols, carbohydrates and a volatile oil¹². Specifically, the extract of Achillea santolina contains chemical constituents such as santoflavone, artemetin, α- santonin, β- sitosterol, lupeol, and leukodin¹¹ that exhibit cytotoxicity¹³, anti-inflammatory¹⁴, antimicrobial¹⁵, cholesterol level reduction^16,17,antioxidant¹⁸ and anticancer activity¹⁹, respectively.

Lately, there has been a great interest in the therapeutic potential of medicinal plants that may be due to their phenolic content, specifically flavonoids^20,21. Flavonoids make up a wide range of biologically active compounds, many of them are incorporated as components in different medicinal preparations over thousands of years to cure various human diseases. Flavonoids are widespread within the plant kingdom; they are the most prevalent pigments next to chlorophyll and carotenoids ²².

Chemically, flavonoids are made up of fifteen carbon skeleton consist of A and B benzene rings linked through a heterocyclic pyrane ring (C). Flavonoids are divided into various classes such as flavones (e.g., flavone, apigenin, and luteolin), flavonols (e.g., quercetin, kaempferol, myricetin, and fisetin), flavanones (e.g., flavanone, hesperetin, and naringenin), and others. These classes differ in the level of oxidation and pattern of substitution of the C ring, while individual compounds within a class differ in the pattern of substitution of the A and B rings ²³. Flavonoids generally found in plants as glycosylated derivatives and their physiological roles in the ecology of plants are diverse²².

As a dietary constituent, flavonoids show health promoting properties as a result of their high antioxidant activity both in vivo and in vitro systems^24,25. Many diseases are developed or substantially aggravated due to continuous chronic inflammation due to the presence of free radicals. Flavonoids functional characteristics fight many inflammatory processes underlying many chronic ailments such as cancer, obesity, and neuroinflammation²⁶.

For all of these beneficial effects, estimating the yield weight, total flavonoids content and the concentrations of proposed individual flavonoids present in Iraqi Achillia santolina aerial parts are of great value. According to the best of authors’ knowledge, this is the first qualitative and quantitative flavonoids profile of this naturally grown plant in Iraq.

MATERIAL AND METHODS:

Plant Collection and Classification:

The grown plant of Yarrow (Achillia santolina L.), known locally as Qaysoom Al-Mukadis , was collected from Karbala Governorate located in central part of Iraq between July-August/2020. The plant was identified andauthenticated by Assistant Professor Dr. Sukaina Abbas at the Department of Biology, College of Science/University of Baghdad. The aerial parts of the plant were washed, dried in the shade for 14 days^27,28 and grinded using an electric mill. The pulverized parts were stored in a clean, dry glass container till use.

Extraction of Total Flavonoids:

Forty grams of powdered aerial parts were macerated in hexane in 1: 10 ratio²⁹ for 48 hours to remove non-polar material. The plant macerate filtered using Whatman paper No.1 and solvent residue was allowed to evaporate and weighed.

The defatted marc material was extracted by reflux condenser apparatus with 10% hydrochloric acid till exhaustion in a (1:5) plant to solvent ratio. This process repeated twice with fresh solvent. The solution was allowed to cool and then filtered. The filtrate was poured into a separating funnel and the flavonoids were partitioned repeatedly with ethyl acetate three times. The gathered organic layer was washed with distilled water to get rid of the excess acid then dried over anhydrous sodium sulfate , filtered, evaporated to dryness by rotary evaporator at a temperature of 45^ᵒC (Heidolph), weighed and stored at -20^ᵒC till further investigations³⁰.

Qualitative analysis of Total Flavonoids by Thin Layer Chromatography Technique (TLC):

Thin layer chromatography (TLC) was used for the identification of the constituents of the ethyl acetate fraction.³¹

Stationary phase:

Premade silica gel GF_254nm plates (20x20cm) of 0.25mm thickness were used and activated by an oven at 110 C° for 30 minutes before use.

Standards preparation:

Standards used were prepared by dissolving 1 mg of each standard in 1ml of methanol to obtain a concentration of 1mg/ml.

Sample Preparation:

One mg ethyl acetate fraction residue was dissolved in 1 ml of methanol to obtain concentration of 1mg/ml for use in TLC.

Developing Solvent system:

For the identification and detection of ethyl acetate (EA) fraction constituents, several mobile phases were prepared. The mobile phase with best separation properties was chosen.

S1= Ethyl acetate: formic acid: glacial acetic acid: water (100:11:11:26 v/v^)32-34

S2 =Toluene: ethyl acetate: formic acid (50:40:10 v/v)³⁵

S3= Toluene: ethyl acetate :formic acid (30:70:10, v/v) ³⁶

S4= Ethyl acetate: n-butanol: formic acid: water (50:30:10:10, v/v)³⁷

S5=Toluene: dioxane: glacial acetic acid (90:25:4 v/v)³²

S6= n-hexane: ethyl acetate: acetic acid (62:28:10 v/v)³⁸

S7= Modified mobile phase n-hexane: Ethyl acetate: acetic acid (42:48:10 v/v) {Researchers}

S8= Modified mobile phase n-hexane: Ethyl acetate: acetic acid (52:38:10 v/v) {Researchers}

One hundred milliliters of mobile phase was poured into a chromatographic jar (23×23cm×7cm), covered and allowed to stand for 45min before use to ensure saturation. A drop of each of the ethyl acetate fraction and reference standards (1mg/ml) were placed on the TLC plates using capillary tubes, allowed to dry, and then developed by ascending technique. Plates were inspected after development under UV light at 254nm and 366nm. The separated spots were marked with a pencil and the R_f value (retention factor) for each constituent visualized as a fluorescent spot under UV light was calculated.

(R_f value = Distance moved by the constituent/Distance moved by the mobile phase)

Qualitative and Quantitative analysis of EA fraction by RP-HPLC^39,40

RP-HPLC ( reversed phase-High performance liquid chromatography) was employed for its high sensitivity and accuracy to confirm the TLC results and to estimate individual flavonoids in EA fraction.

Samples and standards preparation:

The samples and standards (Apigenin, Kaempferol, Quercetin, Luteolin and isorhamnetin) were prepared in a concentration of 1mg/ml by dissolving 1 mg of each standard and sample in 0.5ml HPLC-grade methanol, and sonicated for 10 minutes and volume completed to 1 ml by solvent system (acetonitrile and 1% aqueous acetic acid 1: 9). All working solutions were filtered using 0.45μm Millipore filter and the mobile phase was degassed prior to solutions injection.

RP-HPLC conditions

The separation and detection of flavonoids performed by using Binary high pressure gradient pump (P6.1L), Diode array detector (DAD 2.1L), Sample loop (20µl) and injector (D1357) (Germany), Analyses and system control software (Claritychrom, V 7.4.2.107) Data apex, (Czech Republic). The separation on C₁₈ column (250 × 4.6mm i.d., 5µm particle size, 80 Å pore size) (Knuaer, Germany). Solvent (A) consist of 1% aq. acetic acid solution and (Solvent B) consist of acetonitrile. The flow rate was adjusted to 1ml/min and the column temperature was set at 28⁰C and the injection volume was kept at 20μL. A gradient elution was carried out by changing the proportion of solvent B to solvent A. The gradient elution was changed from 10% to 40% B in a linear approach for a period of 28 min, from 40 to 60% B in 39min, from 60 to 90% B in 50 min. The mobile phase composition back to initial condition (solvent B: solvent A: 10: 90) in 55 min. HPLC chromatograms were detected using a photo diode array UV detector at wavelengths (272, 280 and 310nm). Flavonoids detection was based on matching retention time and absorbance spectrum of the standards. The concentrations of proposed individual flavonoids were estimated by preparing serial dilutions (0.1-150μg/ml) of external standards to establish calibration curve between concentrations and their equivalent peak area. The procedure repeated three times and calculated as mean ±SD.

Quantitative estimation of Total Flavonoids content (TFC) of EA fraction by colorimetric assay:

To estimate TFC in ethyl acetate fraction, Quercetin was employed to construct a calibration curve in concentrations of (25, 50, 80, 100, 150, 200, 250 and 300mg/100ml) in 80% ethanol (v/v). An aliquot of (0.5) ml of the standard’s dilutions and sample were mixed with 1.5ml 95% ethanol (v/v), 0.1ml 10% Al(NO₃)₃, 0.1 ml of 1mol/l potassium acetate and 2.8 ml distilled water in different test tubes. For the blank, the volume of 10% Al(NO₃)₃ was substituted by the same volume of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured by UV-Vis spectrophotometer at 415 nm. The assay performed in triplicates and the flavonoid content was calculated as mean ± SD and expressed as mg/g of Quercetin equivalent (QE) of dry extract^41,42.

RESULTS AND DISCUSSION:

Concerning the percentage yield, the weight of the EA residue was 0.7689 g / 40 g dry plant materials and in turn the percentage yield (1.922%) was calculated according to the following: ⁴³

Percentage yield= [W1-W2/ W3] x 100

W1: Weight of the container and dry residue, W2: Weight of the empty container, W3: Weight of the plant material

Many literatures have reported several factors may influence the extraction yield such as concentration and type of the solvent⁴⁴, temperature, extraction time, plant part and solvent to plant ratio.

The presence of flavonoids as glycosides in the plant, restrict their quantification. Therefore, hydrolysis should be performed to free aglycons by reflecting the plant material with aqueous HCl, which is then quantified by different methods. In addition, low PH of the solvent enhances extraction efficiency of flavonoids as reported in literature suggesting flavonoids recovery increased in acidic pH (2.5–3.5) and decreased at higher pH^45,46.

Qualitative analysis of Total Flavonoids by TLC:

Components of the EA fraction were identified using analytical TLC as it is a quick and cost-effective technique^47,48. The principle of TLC is based on differences in the affinity of a compound to mobile and stationary phases, and this influence the rate at which it migrate^46-49.TLC remain the technique of choice to obtain the first distinct finger print of active components found in medicinal plants and herbal drugs^46-49. The identification was based on similarities in R_fvalues of separated compounds and standards.

Ethyl acetate fraction developed in different solvent systems designated as (S1-S8). All solvent systems showed poor separation except for (S7), our modified mobile phase which was the most appropriate for the separation of fraction’s components based on trial and error. The components of EA fraction were separated and identified according to their corresponding standards as shown in Figure (1) and Table (1).

A=Apigenin, L=Luteolin, K=Kaempferol, Q=Quercetin, PCA=p-Coumaric, and EA sample= the ethyl acetate fraction

Figure 1: TLC chromatogram of EA fraction of Iraqi A. santolina developed in (S7) modified solvent system with standards detected under UV 254 nm.

Table 1: R_fvalues of separated components of EA fraction with corresponding standards values by TLC

Standards	R_fvalue of standard	R_fvalue of EA components
p-coumaric acid (PCA)	0.825	0.822
Kaempferol (K)	0.822	0.820
Apigenin (A)	0.735	0.722
Quercetin (Q)	0.677	0.670
Luteolin (L)	0.593	0.580
Ethyl acetate fraction (EA)	Contain all above spots with other unknown spots

The qualitative and quantitative analysis of EA fraction components were performed by RP-HPLC for its high sensitivity and accuracy. The EA fraction components were identified by matching retention time and absorbance spectrum with those of standards. The detected flavonoids were Kaempferol (29.2 min), Isorhamnetin (38.7 min), Luteolin (39.8 min), Quercetin (41.6 min) and Apigenin (44.1min) as shown in Figure2.

Figure 2: RP-HPLC chromatogram of phenolic compounds in EA fraction

PCA=p-coumaric acid, K= Kaempferol, Iso= Isorhamnetin, L=Luteolin, Q= Quercetin, A=Apigenin

Quantitative analysis of ethyl acetate (EA) fraction by colorimetric method:

Total flavonoids have most commonly been detected by colorimetric methods, such as Al (NO₃)₃ assay. Quantitative determination of total flavonoids of the EA fraction was done on the basis of linearity of Quercetin calibration standard curve (y = 0.0073x) (R² = 0.9955) achieved with (25-300mg/100ml) concentrations (Figure3). TFC in EA fraction of aerial parts of Iraqi A. santolina was estimated (22.01362 ± 2.1 mg / g) as Quercetin equivalent of dry extract.

Figure 3: Calibration curve of Quercetin standard for estimation of total flavonoids content in EA fraction of aerial parts in Iraqi Achillia santolina

The results revealed very close to considerable differences between TFC of the current study and other studies. One study conducted by Salem et.al. showed a TFC of A. santolina leaves and flowers collected from Mersa Matruh (Egypt) of (24.66±1.97mg/g) expressed as rutin equivalent dry weight⁵⁰, while another study carried out by Ardestani and Yazdanparast⁵¹ showed TFC of aerial parts of A. santolina was (49.04±1.98) expressed as mg catechin equivalent/g dried extract. The variability in content of flavonoids among the studies may be influenced by the flavonoid standard employed in the quantification process⁵², the plant part used in extraction, geographic origin of the plant, growing conditions or material preparation that may affect the content, such as harvest time and post harvest preparation employed (e.g., fresh preparation, fermentation, acidification, and type and extent of thermal processing) environmental, seasonal and year-to-year variations. These factors have been described to crucially affect the content and composition of flavonoids.^53-58.

Quantitative estimation of individual flavonoids of EA fraction:

The concentrations of individual flavonoids were calculated according to straight line equation of calibration curve for each standard as in Figures 4. Luteolin was the highest followed by Apigenin, Isorhamnetin, Quercetin, and Kaempferol with concentrations of (0.223±0.17), (0.184±0.18), (0.151±0.14), (0.148±0.08), and (0.024±0.10)μg/mg of dry extract, respectively.

Figure 4: Calibration curves of Apigenin, Luteolin, Quercetin, Isorhamnetin, Kaempferol

Valuable medicinal plants depend on the bioactive constituents and their quantity. A principal phytochemical constituent of Iraqi Achillia santolina are flavonoids. Flavonoids are known to have antioxidant effects and have been shown to impede the development and progression of tumors⁵⁸; decrease in coronary heart disease has been related to the intake of flavonoids⁶⁰. Apart from the antioxidant characteristics it possesses, flavonoids show other functions such as protection against platelet aggregation, microorganisms, hepatotoxins, viruses, tumors, ulcers, free radicals, inflammation, and allergies⁶¹.

Moreover, the estimation of yield value reflect the optimal conditions for extraction, while the total flavonoid content and individual flavonoids concentrations considered as quality guide for the plant and may correlate with the efficacy of biological activities.

CONCLUSION:

Reviewed literature showed few studies deal with the crude extract of Achilia santolina grown in Iraq but there were no studies found concerning the yield value, total flavonoids content and concentrations of individual flavonoids in EA fraction. The investigational results of the current study showed Iraqi Achillia santolina is a valuable plant and considered as a source of flavonoids for potential treatment of many diseases. To the best of our knowledge, this study is the first.

CONFLICTS OF INTEREST:

There are no conflicts of interest.

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Received on 30.03.2022 Modified on 22.05.2022

Research J. Pharm. and Tech 2023; 16(1):287-293.

DOI: 10.52711/0974-360X.2023.00052