Phytotherapeutic Potentials of Synedvilla Nodiflora: In-Vitro Quantification of Phytochemical Constituents, Antioxidant Capacities and Skin Enzymes Inhibiting Activities

Quantification of Constituents, Capacities and Skin Enzymes Inhibiting Abstract Synedrella nodiflora is a useful medicinal plant that has been evaluated for its application in treatment of several ailments in sub-Saharan Africa and Bangladesh since time immemorial. In the present study, the methanol extract of the plant was investigated in-vitro for the presence of some essential phytochemicals, its antioxidant capacities, activity inhibition of skin degenerating enzymes of elastase and tyrosinase as well as its anti-lipid peroxidation activity. Determination of the total antioxidant capacities of the extract was achieved through evaluation of oxygen radical absorbance capacity (ORAC), ferric ion reducing antioxidant potential (FRAP) and iron-II induced inhibition of lipid peroxidation (LPO) assays. The quantitative analysis outcome of this study showed the relative abundance and concentration of flavanol, alkaloid, flavonol, phenolics, tannin, proanthocyanidins and saponin in Synedrella nodiflora methanol extract with the relative abundance (%) of the phytochemicals is in the order of polyphenols (19.09)>Saponin (18.61)>flavonol (18.00)> proanthocyanidins (14.52) > flavanol (10.35)>Alkaloid (10.07)>tannins (9.36). The outcome of this study showed the ability of the plant extracts to scavenge free radicals and to inhibit degradation of lipids due to oxidative damage. Thus, S. nodiflora may be used in the treatment and management of oxidative stress and its related diseases. It was also observed that the plant extract possess a mild in-vitro tyrosinase and elastase inhibiting activities which implies that the plant may find applications in cosmetic preparations for skin depigmentation and anti-wrinkle agents in


cancer etc. Such plants include Citrulus colocynthis, Acacia arabica,
Ocimum gratissimum, Azadirachta indica, Phyllanthus amarus, Eclipta alba and Vernonia amygdalina [8][9][10][11][12][13]. The ethnopharmaceutical and ethnomedicinal applications of these plants are due to their varying degree of bioactive phytochemical constituents as well as their characteristic antioxidant properties [14][15][16][17]. The enzymes inhibiting properties of medicinal plants have been found exciting, adding more value to phytotherapy and making plants an excellent alternative to conventional synthetic chemicals in biomedicine. The alpha amylase and alpha glucosidase inhibition activities of some plants have been reported [18,19]. Recent independent researches by [20,21,22] have shown the inhibitory effects of some plant materials on the enzyme tyrosinase present in the skin and they Synedrella nodiflora [23]. Synedrella nodiflora is a branched ephemeral herb that can grow up to 80 cm tall. It is characterized with a shallow root system which is usually strongly branched. The lower part of the stem may root at the nodes, especially in moist conditions while the leaves exist in opposite pairs and are usually 4-9 cm long. The presence of various bioactive constituents in the leaf extract of Synedrella nodiflora was reported after screening studies by [24,25]. Solvent extracts of Synedrella nodiflora have been shown to possess flavonoids, alkaloids, glycosides, steroids, tannins, saponins and phytosterols, triterpenoids gums and reducing sugars [26][27][28][29]. The plant is essential for the treatment of various diseases and its leaves are eaten as a vegetable by some livestock and human. It also possesses sex hormone activity [30]. The leaves can be used as Pregnant Mare Serum Gonadotrophin supplier in animal husbandry and to improve reproductive parameters in female animals [31].
Literatures on the skin enzymes inhibitory activities of the plant are still limited according to SciFinder and Dictionary of natural products. This research is therefore directed at investigating the inhibitory actions of Synedrella nodiflora on the degenerating actions of enzymes present in the surface of the skin (tyrosinase and elastase) as well as its phytochemical constituents and antioxidant properties to support and validate its acclaimed ethnopharma-cological application.

Chemicals and Reagent
The reagents and standards used for this work were all of analytical grades with high percentage purity, secured from Sigma-Al-

Samples Collection and Preparation
Aerial part of S. nodiflora was sourced from a local farm in Ikere-Ekiti (7.4991° N, 5.2319 ° E), Ekiti State Nigeria. The plant was identified by the Herbarium curator at the Department of Biological Science Koladaisi University, Ibadan, Nigeria, where a voucher specimen number Kdu/IB/034 was assigned to the S. nodiflora.
The obtained plant materials were prepared by washing them with distilled water and drying at room temperature for two weeks.
The plant parts were crushed separately using pestle and mortar.
Thereafter, the crushed parts were mixed together, pulverized by an electric blender into a homogenized powder, weighed and stored in different airtight sterile sample bottles pending analysis.

Extraction
The powdered plant material was extracted with methanol. Approximately 50g of the powdered material was soaked in 1000 mL of the solvent in a vial for 72 hours for cold extraction. The extract was filtered and concentrated at 50 0 C using rotary evaporator. The concentrate was stored in an air tight sample vial pending analysis.

Determination of Total Phenol
The total phenolic content of the aerial part of the plant was determined using the Folin-Ciocalteu method [32]. The procedure involved adding both distilled water and Folin-Ciocalteu reagent to a 125 μL of the solvent extract. The mixture was allowed to stand for 6 min before the adding sodium carbonate solution (7.0% w/v).
Thereafter, the mixture was allowed to stand for 90 min after which the absorbance was read at 760 nm on a SpectrumLab70 spectrophotometer and the result were expressed in terms of Gallic acid in mg/mL of extract.

Determination of Saponin
The saponin concentration of the plant was determined using spectrophotometric method as described by [33]. In this method, 2.0 g of the extract was weighed into a beaker containing isobutyl alcohol (butan-2-ol) was added. The mixture was stirred and filtered through No 1 Whatman filter paper into a beaker containing

Determination of Tannin
The total tannin content was assessed by the experimental protocol of [18] with slight modifications. The tannin determination was carried out as follows. Approximately 0.5 g of the extract was diluted with 90% ethanol. 0.1 mL of the diluted sample was measured and added to 2 mL of Folin Ciocalteu reagent. After 10 min, 7.5 mL of sodium carbonate (7%) solution was added and the mixture incubated for 2 hours. The absorbance of this mixture was measured at 760 nm and the tannin content was estimated using tannic acid (TA) curve as the standard.

Determination of Alkaloid
Alkaloid content of the plant's solvent extract was determined by weighing 5.0 g portion of the solvent extract into a beaker and 250 mL of 10 % acetic acid in ethanol was added and allowed to stand for 5 min. The mixture was filtered and the extract was concentrated on a water bath to one fifth of the original volume. Ammonium hydroxide solution was then added in drops to the concentrated extract until the precipitation was completed [19]. The precipitate was collected, washed severally with dilute ammonium hydroxide and filtered. The residue was dried in a desiccator and weighed.

Flavanol Content
The flavanol content of the S. nodiflora was determined spectrophotometrically in accordance to the modified method of Popoola et al. [7]. A 25 mL 0.05% 4 dimethylaminocinnam-aldehyde (DMA-CA) solution prepared by dissolving DMACA in 8% HCl prepared in methanol was added to 50 mg of the solvent extract. The mixture was allowed to stand for 30 min and the absorbance was read on a SpectrumLab70 spectrophotometer at 640 nm. The result was expressed in terms of catechin equivalents in mg CE/mg of extract.
The determination was carried out in triplicate.

Determination of Flavonol
The flavonol content of the plant extract was determined by the method described (2008) with slight modifications. 2 mL of AlCl3 prepared in ethanol and 3 mL of 50 g/L sodium acetate solution were added to 2 mL of the extracted sample in a test tube and mixed.
The mixture was incubated for three hours (3 hrs) at 20 oC. Series of stock solution of 20, 40, 60, 80, and 100 μg/mL were thereafter prepared. The absorbance of the solutions was measured at 440 nm against a blank at 593 nm using a SpectrumLab70 spectrophotometer. The total flavonol content was calculated in terms of quercetin equivalent in mg QE/mL of sample from the calibration curve.

Determination of Proanthocyanidins
The total proanthocyanidins content of the plant material was determined by the procedure of [45]. An amount 0.5 g of the meth-anol extract of the plant material was vortex mixed with 3 mL of vanillin (4 %) prepared in methanol and 1.5 mL of hydrochloric acid. The mixture was allowed to stand for 15 min at ambient temperature. Thereafter, the absorbance was read at 500 nm. The total proanthocyanidin content was expressed in terms of catechin (CE mg/g).

Ferric Reducing Antioxidants Potential (FRAP) Assay
The FRAP assay was performed according to a modified method of [19]

Inhibition Lipid Peroxidation Assay (LPO)
For the determination of the lipid peroxidation inhibition assay, a little modification was made on the adopted method of Snijman

Oxygen radical absorbance capacity (ORAC) assay
The methods of [22,35] were adopted for the ORAC assay: The 538 nm emission wavelengths. The ORAC value was calculated by dividing the sample curve-area by the trolox curve area [22]. The ORAC value of the extract was expressed as mg/mL Trolox equivalent (TE) of dry extract.

Enzymes Inhibition Activities Tyrosinase Inhibition Assay
The aerial part of the S. nodiflora was also assayed for its tyrosinase inhibition activity in a 96-well reader using a modified procedure of [36]. The anti tyrosinase activity of the plant was carried

Elastase Inhibition Assay
The Elastase inhibition activity of the plant material was assayed by modifying the method of [37]. In this method, N-succ-(Ala)3-nitroanilide (SANA) was used as the substrate the release of p-nitron-

Results and Discussion
The phytochemical analyses of the S. nodiflora extract revealed the relative abundance ( Figure 1) and concentration (Figure 2 These compounds are often referred to as powerful chain-breaking antioxidants [39][40][41]. The presence of these bioactive phytochemicals in the plant showed that the plant is a useful antioxidant material that may exert protective effects against inflammation, cancer diabetes and cardiovascular disease; they may also play a vital role in microbial inhibition.   Further, the results of the present study demonstrate that the plant exhibit variations in its phytochemical constituents (Table 1). Polyhenols (44.97 ± 1.33 mg of GAE/mL) was the most abundant  Table 1). The mechanism of lipid peroxidation has been suggested to proceed via a free radical chain reaction Zhiyong and Yuanzong [42] which has been associated with cell damage in biomembranes [22,43]. The damage has been shown to initiate the development cancerous cells, cardiovascular diseases and diabetes according to [44,45]. The potency of the S. nodiflora extract to inhibit the process of lipid peroxidation was investigated in this study. The methanol extract of the plant was able inhibit lipid peroxidation by 55.60% well below that of the standard EGCG (90.95%). However, the value (Table 2) recorded for this plant showed its mild ability to inhibit lipid peroxidation processes ( Table 2). The reducing power of a compound is related to its electron transfer ability and may therefore serve as a significant indicator of its potential antioxidant activity [7]. To estimate the reductive ability of the plant's extract, the ferric ion reducing antioxidant potential of the extract was assayed. The result of this study showed Fe 3+ to Fe 2+ transformation potential of the plant's extract with FRAP value of 681.10±0.13 mg/ mL which is higher than that of the standard 3.20±0.01 mg/mL as shown in Table 2. The higher reducing ability of the plant's extract implies a higher electron donating ability of the extract.    Table 3).
The inhibitory effect of S. nodiflora on tyrosinase activity was investigated in-vitro and the result is presented in Table 3. The assay was carried out to show the possibility of using the plant to solve problems relating skin pigmentation. The potential of the plant's extract and kojic acid (standard) to inhibit this enzyme increased from 20 to 100μg/mL with respect to the results obtained from this study. This shows that the inhibition activity of the materials increased with increasing concentration of the extract (Figure 3,4).
The S. nodifora extract and kojic acid showed optimum inhibition (39.75 and 95.9 % respectively) at the concentration of 100 μg/ mL (  The tyrosinase inhibition activity of the plant showed its value as a promising material for solving the problems of hyperpigmentation or may be used for depigmentation purpose. Elastase inhibition activity of the S. nodiflora was also investigated to evaluate its applicability in skin treatment and problems solving involving skin wrinkling and dry skin. The performance efficiency of the plant's extract to inhibit Elastase at optimum concentration (100μg/mL) was 44.4%. The percentage by which both the extract and the standard (xanthone) inhibited the enzyme increase with their increasing concentrations respectively. It is possible that increasing the concentration of the extract beyond 100μg/mL could increase its inhibition activities. However, the plant material was able to demonstrate ability to maintain healthy skin and anti-wrinkle activities even at lower concentrations.

Conclusion
The presence of free radicals and reactive oxygen species is a major cause of skin aging and cellular oxidative stress related prob-