Phytochemical analysis, total flavonoid, total phenolic contents and ferric reducing power of extracts from leaves and stem bark of Buddleja salviifolia

Chloroform, ethyl acetate and methanolic extracts from leaves and stem bark of Buddleja salviifolia were prepared. The phytochemical profiles, total flavonoid contents (TFC), total phenolic contents (TPC) and ferric reducing power of these extracts were studied. The presence of alkaloids, tannins, phlobatannins, saponins, sterols, flavonoids, coumarins, phenolics, terpenoids, quinones and reducing sugars were identified from these extracts. The TFC of these extracts was found to be in the range of 11.36±0.98 to 30.76±1.15 mg QE/g DW, respectively. The TPC of these extracts was found to be in the range of 175.00±0.41 to 465.27±2.65 mg GAE/g DW, respectively. Additionally, these extracts showed a weak to moderate ferric reducing power compared to ascorbic acid. B. salviifolia finds therapeutic applications in traditional medicine. Further studies are required to commercialize products from this plant.


Introduction
Buddleja salviifolia belongs to the Scrophulariaceae family of the Buddleja genus (Van Wyk and Van Wyk, 1997;Ream, 2006;Tank et al., 2006;Van Laere et al., 2011). B. salviifolia is also known by other names such as sailehout, sagewood, wildsaile and lelothoane (Van Wyk and Van Wyk, 1997;Kose et al., 2015). B. salviifolia is a semi-evergreen shrub (Ream, 2006) or trees of 3-8 m height (Drummond, 1981) and is widely distributed in the Southern Africa (Palmer, 1997). B. salviifolia finds therapeutic applications in the traditional medicine in the African continent. Particularly, the leaves of B. salviifolia possess most of the therapeutic potentials and have been used to treat TB, herpes, syphilis, cervical cancer, complications associated with pregnancy, coughs, colic, constipation, neurodegenerative diseases, eye infections and to relieve nausea (Pendota et al., 2013;Kose et al., 2015). The antimicrobial and DPPH radical scavenging activities of various extracts from leaves and stem bark of B. salviifolia have previously been reported Pillai et al., 2019). The present study aimed to analyse the phytochemical profiles, total flavonoid contents (TFC), total phenolic contents (TPC) and ferric reducing power of chloroform, ethyl acetate and methanolic extracts from leaves and stem bark of B. salviifolia species collected from the Kingdom of Lesotho, Southern Africa. This is the first report of this kind from the B. salviifolia and particularly, the species gathered from the Kingdom of Lesotho.

Chemicals and Reagents
AR grades (99.5%) of chloroform, ethyl acetate, methanol, benzene, quercetin, gallic acid and trichloroacetic acid were all purchased from Sigma Aldrich. Benedict's reagent, sodium hydroxide, hydrochloric acid and sulphuric acid were purchased from Minema Chemicals. Potassium ferricyanide, ferric chloride, sodium dihydrogen phosphate, magnesium powder, ascorbic acid, trisodium phosphate and potassium mercuric iodide were purchased from Prestige Laboratory Supplies.

Plant materials
The leaves and stem bark of B. salviifolia were collected in August 2018 at the National University of Lesotho, Roma campus. Voucher specimens for leaves

Preparation of extracts
A mass of 98.88 g of powdered leaves was macerated with 500 mL of chloroform for three days with occasional shaking. The resulting solution was filtered using a vacuum filter (ATB, Model: 284965-H, Power: 230 v 3.0 A, 1320/min, 50 Hz) and the extract was concentrated in vacuo. The concentrated extract was then transferred to a pre-weighted clean and dry beaker. The procedure was replicated two times. Finally, the sample was refluxed with chloroform for 10 hrs and the resulting solution was concentrated in vacuo. A mass of 7.48 g of combined chloroform extract was obtained. Similarly, 8.13 and 14.61 g of ethyl acetate and methanolic extracts were obtained, respectively from 100.23 and 175.45 g of powdered leaves. Using the similar extraction procedure, 1.07, 2.18 and 8.86 g of chloroform, ethyl acetate and methanolic extracts were obtained, respectively from 200. 09, 200.01, 202.53 and 157.82 g of powdered stem bark.

Detection of alkaloids
A mass of 5.0 mg of each extract was dissolved in 2.0 mL of distilled water separately and then three drops of Wagner's reagent was added. The formation of a reddish-brown precipitate indicates the presence of alkaloids.

Detection of phenolics
A mass of 0.5 g of each extract was dissolved in 1.0 mL of distilled water separately and was heated in a water bath at 95℃ for 30 mins. The resulting mixture was filtered and 3.0 mL of 5% (w/v) of ferric chloride was added to the filtrate followed by the addition of 1.0 mL of 1% (w/v) potassium ferricyanide. A formation of green colouration indicates the presence of simple phenols and the blue colouration indicates the presence of polyphenols.

Detection terpenoids
A volume of 0.5 mL of distilled was added to 0.25 g of each extract separately and then 1.0 mL of chloroform was added with vigorous shaking and swirling. A formation of reddish-brown colouration on the interface upon the addition of 3.0 mL of concentrated sulphuric acid to the mixture indicates the presence of terpenoids.

Detection of flavonoids
A mass of 0.50 g of each extract was dissolved in 2 mL of methanol and heated in a water bath separately. A small amount of magnesium powder was added to the mixture and few drops of concentrated hydrochloric acid were added thereafter. A formation of dark brown colouration which gradually shades to deep red or pink colouration indicates the presence of flavonoids.

Detection of sterols
A mass of 0.3 g of each extract was dissolved in 2.0 mL of chloroform separately and then filtered off. A volume of 1.0 mL of concentrated sulphuric acid was added to each filtrate. A formation of two phases of which the chloroform phase turns red indicates the presence of sterols.

Detection of saponins
A volume of 2.0 mL of distilled water was added to 0.1 g of each extract separately and the contents were agitated in a test tube for 15 mins. A formation of a 1 cm layer of foam indicates the presence of saponins.

Detection of tannins
A mass of 0.2 g of each extract was dissolved in 2.0 mL of distilled water separately and heated in a water bath at 95℃. Upon filtration, few drops of concentrated sulphuric acid were added to each filtrate followed by the addition of a few drops of 5% (w/v) ferric chloride. A deposition of blue, black, green or blue-green precipitate indicates the presence of tannins.

Detection of phlobatannins
A mass of 0.1 g of each extract was boiled in 3.0 mL of 1% aqueous hydrochloric acid separately. A formation of a red precipitate indicates the presence of phlobatannins.

Detection of quinones
A mass of 0.25 g of each extract was dissolved in 0.5 mL of distilled water separately and then 1.5 mL of concentrated hydrochloric acid was added to it. A deposition of green or white precipitate indicates the presence of quinones.

Detection of reducing sugars
A mass of 0.1 g of each extract was dissolved in 2.5 mL of distilled water separately. A volume of 2.0 mL of the extract solution was mixed with 2.0 mL of Benedict's solution. Each of these mixed contents was heated in a boiling water bath at 95℃ for 5 mins or until there was an observable colour change. A formation of brick-red colouration indicates the presence of reducing sugars.

Detection of coumarins
A mass of 0.3 g of each extract was dissolved in 1.0 mL of distilled water separately and then 1.0 mL of 10% (w/v) sodium hydroxide and 1.0 mL of chloroform were added. A formation of yellow colour indicates the presence of coumarins.

Determination of total flavonoid contents (TFC)
The TFC of each of these six extracts was evaluated separately using the aluminum chloride calorimetric method as described in the literature (Joshi et al., 2019). The results were calculated based on a calibration curve plotted using quercetin. A stock solution of quercetin was prepared by dissolving 25 mg of quercetin in 1.0 mL of methanol and serial dilutions of 25, 20, 15, 10 and 5 µg/mL were prepared from the stock solution. A solution of 10 mL of each extract in methanol at a concentration of 1000 µg/mL was prepared separately. Aliquots of 3.0 mL of each extract or quercetin were mixed separately with 2% (w/v) aluminum chloride in clean and ovendried test tubes. The experiment was performed in triplicates and the contents were incubated at room temperature for 15 mins. The absorbance was measured at 420 nm using MRS Spectro UV-11 spectrophotometer. The total flavonoid contents of each of these extracts were estimated from the quercetin calibration plot and are expressed as milligrams of quercetin equivalents per gram of extract dry weight (mg QE/g DW).

Determination of phenolic contents (TPC)
The TPC of each of these six extracts was determined separately using Folin-Ciocalteau assay (Kokate, 1994) with slight modifications. The results were calculated based on a calibration curve plotted using gallic acid. Briefly, 1000 µg/mL of stock solution of gallic acid was prepared by dissolving 10.0 mg of gallic acid in 10 mL of methanol. Two-fold serial dilutions of 1000 to 15.625 µg/mL were prepared from the stock solution. A solution of 10 mL of each extract in methanol at a concentration of 1000 µg/mL was prepared separately. Aliquots of 2.0 mL of each extract or gallic acid solution were mixed separately with 1.0 mL of 1% (v/v) Folin-Ciocalteau reagent in clean and oven-dried test tubes. The reaction mixture was allowed to stand for 4 mins and then 1.0 mL of 0.7M sodium carbonate was added. The content was shaken vigorously for 15 s then incubated in the dark for 25 mins at room temperature with occasional agitation. The experiment was performed in triplicates. The absorbance of the reaction mixture was measured at 765 nm using MRS Spectro UV-11 spectrophotometer. The total phenolic content of each extract was estimated from the gallic acid calibration plot and are expressed as milligrams of gallic acid equivalents per gram of extract dry weight (mg GAE/g DW).

Evaluation of ferric reducing power
The ferric reducing power of these six extracts was determined using a method described in literature (Fejes et al., 2000) with slight modification. The ability of an extract to transform Fe (III) to Fe (II) has been termed as its ferric reducing power (Fejes et al., 2000;Saeed et al., 2012). The formation of Prussian blue colour indicates the transformation and the measure of absorbance at 700 nm indicates its ferric reducing power (Saeed et al., 2012). Briefly, a stock solution of 0.2 mg of each extract in 1 mL of methanol was prepared, separately. Two-fold serial dilutions of 200 to 0 µg/mL were prepared. The 0 µg/mL solution served as negative control while ascorbic acid served as a positive control. Aliquots of 2.0 mL of each extract at various concentrations were mixed with 2.0 mL of 0.2M phosphate buffer (pH 6.6), 2.0 mL of 0.01% (w/v) potassium ferricyanide solution and the mixture was separately incubated at 50℃. A volume of 2.0 mL of 0.1% (w/v) trichloroacetic acid was added to the mixture and the content was centrifuged at 3000 rpm for 10 mins. The upper layer was collected thereafter and aliquots of 2.0 mL of the supernatant liquid were mixed further with 0.4 mL of 0.1% (w/v) freshly prepared ferric chloride solution and 2.0 mL of distilled water. The absorbance (optical density) of the content was measured at 700 nm using a spectrophotometer (MRS Spectro UV-eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER 11) after allowing the reaction to occur for 10 mins. Ferric reducing power is a measure of absorbance in a reaction and the higher absorbance indicates the higher the reducing power of an extract or pure compound (Saeed et al., 2012).

Statistical analysis
All determinations were performed in triplicates (n = 3) and the results are expressed as mean±SD. Data analysis was performed using SPSS 17.0 application software by means of two-way analysis of variance (ANOVA). The means were considered statistically significant when p≤0.05.
However, all three leaf extracts (1-3) and all three stem bark extracts (4-6) showed lower ferric reducing power compared to a positive control (refer to Table 2 and Figure 3). This is the first report on the preliminary phytochemical screening of various extracts from B. salviifolia.
However, terpenoids have previously been reported from other species of the Buddleja genus (Houghton et al., 2003). In a previous report, the TFC of dichloromethane/methanol (1:1) and water extracts from whole plants of B. salviifolia have been reported as 23.95±0.11 and 12.11±0.26 mg QE/g DW (milligrams of quercetin equivalents per gram of dry sample), respectively (Adewusi et al., 2011). Similarly, the TPC of these two extracts has been reported as 169.66±0.33 and 77.92±0.91 mg TAE/g DW (milligrams of tannic acid equivalents per gram of dry sample), respectively (Adewusi et al., 2011). In the present study, the TFC and TPC of various extracts from leaves and stem bark of B. salviifolia were found to be in the range of 11.36±0.98 to 27±2.65 mg GAE/g DW, respectively. The discrepancy in the TFC and TPC in our study and the previous report may be due to the polarity of different solvents and plant parts used for the extraction of active components. Extracts 2, 3, 5 and 6 showed significant ferric reducing power due to the presence of tannins (refer to Table 1). Tannins have been reported to have ferric reducing power (Okuda, 2005). Various extracts from B. salviifolia have previously been reported to have DPPH radical scavenging activity , which could be due to the presence of phytochemicals such as polyphenols, phenolics and flavonoids. These phytochemicals have been reported as important classes of natural antioxidants (Array et al., 2019;Rodrigues et al., 2019).

Conclusion
The phytochemical profiles, TFC, TPC and ferric reducing power of chloroform, ethyl acetate and methanolic extracts from leaves and stem bark of B. salviifolia were studied. The presence of alkaloids, tannins, phlobatannins, saponins, sterols, flavonoids, coumarins, phenolics, terpenoids, quinones and reducing sugars was identified from these extracts. The TFC of these extracts was found to be in the range of 11.36±0.98 to 30.76±1.15 mg QE/g DW, respectively. The TPC of these extracts was found to be in the range of 175.00±0.41 to 465.27±2.65 mg GAE/g DW, respectively. Additionally, these extracts showed a weak to moderate ferric reducing power compared to ascorbic acid. Further studies are required to commercialize products from this plant.