Inhibitory Effect of Bryophyllum pinnatum ( Lam . ) Oken leaf Extract and their Fractions on α-amylase , α-glucosidase and Cholinesterase Enzyme

Bryophyllum pinnatum (Lam.) Oken leaves are employed as food and as traditional medicines. This study investigates the antioxidant activity (reducing power, DPPH, ABTS, FRAP, H2O2 scavenging ability and metal ion chelating), carbohydrate digesting enzymes activity and inhibitory activity of cholinergic enzyme of aqueous extract and fractions (n-hexane, ethyl acetate, n-butanol, residual aqueous fraction) of B. pinnatum leaves were investigated. Results showed that aqueous extract of B. pinnatum exhibited DPPH radical scavenging abilities, iron chelation, hydrogen peroxide scavenging abilities and reducing power (Fe3+Fe2+). B. pinnatum aqueous extract also had considerably α-amylase and α-glucosidase inhibitory activities with IC50 values 149.20 ± 14.44 μg/mL and 126.15 ± 9.76 μg/mL respectively. Our findings indicated that ethyl acetate fraction contained a considerably higher (p < 0.05) amount of total phenolic, flavonoids, total antioxidant, FRAP, metal ion, ABTS and DPPH radical scavenging activity than other solvent fractions. Furthermore, the ethyl acetate fraction elicited a significantly higher (p < 0.05) inhibitory effects on α-glucosidase (IC50 = 70.90 ± 1.23 μg/ml), α-amylase (IC50 = 62.45 ± 1.22 μg/ml), acetylcholinesterase (AChE) (IC50 = 66.75 μg/mL) and butyrylcholinesterase (BChE) (IC50 = 62.97 μg/mL) activities than other fractions. Hence, B. pinnatum leaves were rich in biologically active components; thus, could be employed to formulate new plant-based pharmaceutical and nutraceutical drugs to improve human health.


INTRODUCTION
With the emerging trends in science, the therapeutic properties of plants have got importance role everywhere, attributable to its pharmacologic activities (such as free radical scavengers, anti-ulcer, anti-malarial, anti-cancer, anti-inflammatory, anti-microbial) and nutraceuticals properties. 1 Reports have targeted on beneficial roles of phytochemicals from plants as well as their health-promoting effects.Amid the numerous secondary metabolites with biological activities, phenolics are accountable for the aforesaid biochemical properties. 2 World societies progressively involved on well-being and diet, natural plant products are budding as valuable alternatives aimed at replacing synthetic drugs.On the premise of the aforesaid issues, new plant-based products or Phytomedicine remain thought of as potential resources.
Several native plants are utilized in folklore (traditional) medicine nowadays to treat illnesses and cure wounds. 3One among such therapeutic plants includes Bryophyllum Pinnatum (Lam.)Oken.The medicinal importance of plants paved means for the in depth pharmaceutical production of drugs from its synthetic chemical, utilizing the active ingredients of that particular plant origin. 4Bryophyllum Pinnatum (Lam.)Oken (Crassulaceae) Synonyms: Kalanchoe pinnata (Lam.)Pers.Usually referred to as life leaf and air plant is widely cultivated as a garden ornamental in tropical America, China, India, Australia and tropical Africa.The plant flourishes throughout the Southern part of Nigeria.Bryophyllum Pinnatum (Lam.)Oken, a succulent recurrent herb that cultivates in tropical, subtropical and warm temperate climate zone.It is employed in ethno medicine as a curative plant for the treatment of high blood pressure. 5Literatures searches reported Bryophyllum juice extract as helpful for the cure of kidney stones. 6Although, there's no proof based clinical indication for these uses in modern medicine and so, such usage might prove dangerous and even fatal in some cases.The leaves of the plant have great medicinal value and are used both internally as well as externally.It additionally possesses numerous properties like haemostatic, refrigerant, emollient, mucilaginous, vulnerary, antiinflammatory, 5 disinfectant and tonic.The herb has been used to facilitate the induction of placental delivery in newly born babies within the southern part of Nigeria.The plant additionally encompasses hydrogen cyanide (HCN), Oxalic acid, citric acid, isocitric acid, oxaloacetate and succinic acid.It's also contains a variety of vitamins such as vitamin C, vitamin B 2 , vitamin B 1 vitamin B 3, vitamin B 6 , Cysteine, Casein hydrolysate and amino acids such as glutamic acid, methionine and phenylalanine. 7he herb may be a suitable supply of inorganic elements such as Na, Ca, K, P, Mg, Mn, Fe, Cu, Zn (3).The Bufadienolides which are active components of Bryophyllum Pinnatum possess anti tumorous, 8 antibacterial, 9 and insecticidal actions.Although, some literatures have reported Bufadienolides to be toxic and as like cardiac glycosides toxicity.Edible fruits, herbs and vegetables largely contain high levels of polyphenols that have high antioxidant activity and are useful against stress conditions in the human body, hence presenting vital health benefits.Though, not solely edible plants are beneficial from this point of view, however, additionally non-edible plant materials are mostly examined with this purpose. 10,11,12In this sense, several food supplements and pharmaceutical products have been developed in recent years exploiting plant-derived constituents.Therefore, the biochemical composition of plants ordinarily employed in folk medicines, or from the same species, are being considered to look for new alternatives in the food industry.The main objective of the study here presented was to analyze the antioxidant capacity (free radical scavenging assays, reducing power, metal chelating and DPPH, ABTS assays), and enzyme inhibitory activity (cholinesterases, α-amylase and α-glucosidase) of Bryophyllum Pinnatum (Lam.)Oken leaves from which scarce data is obtainable in the scientific

Plant material and extraction procedure
Fresh leaves of Bryophyllum pinnatum (Lam.)Oken was obtained (January, 2017) at Oja Oba market Ado-Ekiti.Fresh leaves of Bryophyllum pinnatum was identified and documented by a senior plant scientist from the Department of Plant Biology, University of Ilorin, Nigeria and a voucher specimen number was deposited appropriately at the University herbarium.All plant names in this manuscript are arranged according to the modern revision in "The Plant List", and correspond to the good practices in publishing studies on herbal plants, as described by. 13The fresh leaves were air-dried for three weeks and ground to powder using a mechanical blender.To obtain aqueous extracts, 500 g of air-dried leaves were soaked with 2000 mL of water for 48 h. 14Aqueous extracts obtained were lyophilized and stored until use.

Solvent partitioned fractionation of crude aqueous extracts
Aqueous leaves extract of B. pinnatum was (50 g) sequentially extracted with solvents of increasing polarity (hexane, ethyl acetate, n-butanol and water).Aqueous extract was partitioned between n-hexane (2 x 200 mL) and water to obtain n-hexane fraction (HF) and aqueous portion.The aqueous portion obtained was further partitioned by ethyl acetate (2 x 200 mL) to obtain ethyl acetate fraction (EAF) and aqueous portion.The aqueous portion obtained was further partitioned by n-butanol (2 x 200 mL) to obtain n-butanol fraction (BF) and residual aqueous fractions (AF).The yield of the aqueous extract was 25.25g while the yield for n-hexane fraction, ethyl acetate fraction, n-butanol fraction and residual aqueous fractions are 3.26g, 7.74g, 4.5g and 5.5g respectively.

Evaluation of Antioxidant Activities of Aqueous and Fractions of B. pinnatum leaves Determination of Reducing Property
The reducing power of the B. pinnatum leaf aqueous extract and fractions (HF, EAF, BF, AF) was studied by evaluating the capability of the extract or fractions to scale back FeCl 3 solution using the method of. 152.5 mL of extract was mixed with 2.5 mL of 200 mM phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide.Solution was incubated for 20 min at 50 °C in a water bath and 2.5 mL of 10% trichloroacetic acid was added.Resulting solution was then centrifuged for 10 min at 650 g.After that, 5 mL of the filtrate was mixed with an equal volume of water and one mL, 0.1% FeCl 3 .Above-stated process was conjointly applied to a standard ascorbic acid solution, and absorbance was read at 700 nm.The reducing ability was calculated as percentage inhibition.

Evaluation of DPPH Free Radical Scavenging Ability
DPPH scavenging ability of B. pinnatum leaf aqueous extract and fractions (HF, EAF, BF, AF) against DPPH free radical was assessed as delineated by. 17Concisely, suitable dilution of the extract and fractions (1 mL) was mixed with 1 mL 0.4 mM DPPH solution in methanol.The mixture was left within the dark for 30 min and absorbance was read at 516 nm.DPPH radical scavenging assay was afterwards calculated with reverence to the control (Vitamin C was used as a standard for evaluating DPPH activity).

Data analysis
Results were expressed as the mean ± SEM. 23 The level of significance for the analyses was set to p < 0.05.Mean values were analyzed and compared using One way analysis of variance and Student's t test.These analyses were performed using the free software R version 3.1.1. 24

RESULTS
Table 1 shows the results of the total phenolics, total flavonoids, and Vitamin C content, reducing power and trolox antioxidant capacity by aqueous and various fractions of crude aqueous extract of Bryophyllum pinnatum leaves.The result revealed that both the aqueous and all fractions showed an ability to donate electrons to convert Fe 3+ into Fe 2+ 1, with its IC 50 values (93.64 ± 3.42 µg/mL).The result revealed that the aqueous extracts of Bryophyllum pinnatum scavenged free radicals (20-100 µg/ml).Figure 2 shows the metal ion chelating ability of aqueous extract of B. pinnatum leaves.The result revealed that the aqueous extract of B. pinnatum IC 50 (224.17± 15.22 µg/mL) had a high (p<0.05)metal chelating ability.Figure 3 presents the hydrogen peroxide (H 2 O 2 ) scavenging activities of aqueous extracts of B. pinnatum leaves.The extract was found to exhibit NO inhibition activity.As revealed by the results, the aqueous extract of the leaves possessed IC 50 values of (316.88 ± 12.75 μg/mL).The radical scavenging ability of the B. pinnatum leaves extract was consequently evaluated using the reasonably steady ABTS radical and displayed in Figure 4 with IC 50 (94.97± 3.33 µg/ml) value.The results showed that the B. pinnatum leaves extract quenched ABTS radical (20-100 µg/ml).

Estimation of Hydrogen peroxide radical scavenging ability
The capability of the B. pinnatum leaf aqueous extract and fractions (HF, EAF, BF, AF) to scavenge hydrogen peroxide was determined according to the protocol of. 19Hydrogen peroxide (40 mM) was prepared in phosphate buffer (pH 7.4).Extracts and fractions dissolved (20 -100 μg/mL) in distilled water were mixed with hydrogen peroxide solution (0.6 mL, 40mM).Hydrogen peroxide absorbance was determined 10  Quantitative Determination of Total Phenolics, Total Flavonoids, Tannins and vitamin C Quantitative Determination of Total phenolics, total Flavonoids, Tannins and Vitamin.Secondary metabolites and vitamins were quantitatively determined using previ ously described methods. 15,16,18aluation of α-amylase inhibitory activity Alpha-amylase activity was determined concurring to the protocol delineated by. 20A volume of 250 μL of B. pinnatum leaf aqueous extract and fractions (HF, EAF, BF, AF) at totally different concentrations (20-100 µg mL -1 ) was incubated with 500 μL of porcine pancreatic amylase (2 U mL -1 ) in 100 mmol L -1 phosphate buffer (pH 6.8) at 37°C for 20 min.Two hundred and fifty μL of 1 % starch dissolved in 100 mmol L -1 phosphate buffer (pH 6.8) was then added to the mixture and incubated at 37 °C for 1 h.One mL of DNS color was then added to the solution and boiled for 10 min.The absorbance of resulting mixture was read at 540 nm and the enzyme inhibitory activity was calculated as percentage of control sample without inhibitors.All assays were applied in triplicate.

Estimation of α-glucosidase inhibitory activity
Alpha-glucosidase inhibitory activity was assessed in line with the protocol delineated by. 21with small modifications.Briefly, 250 μL of B. pinnatum leaf aqueous extract and fractions (HF, EAF, BF, AF), at different concentrations (20-100 µg mL -1 ), was mixed with 500 μL of 1.0 U mL -1 α-glucosidase solution in 100 mmol L -1 phosphate buffer (pH 6.8) at 37 °C for 15 min.Thereafter, 250 μL of pNPG solution (5 mmolL -1 ) in 100 mmol L -1 phosphate buffer (pH 6.8) was added and therefore the solution was more mixed at 37°C for 20 min.The absorbance of the free p-nitrophenol was read at 405 nm and therefore the inhibitory activity was expressed as percentage of a control sample without inhibitors.

    glucosidase inhibition
A control A sample A contr (%)

Determination of Cholinesterase Activity
Estimation of Acetylcholinesterase (AChE) and butyrylcholinesterase inhibitory activity Inhibitory activity of AChE was evaluated via an adapted colorimetric method as delineated by. 22The AChE activity was assessed in a mixture Pharmacognosy Journal, Vol 10, Issue 3, May-Jun, 2018    Figure 6 shows the percentage inhibitory activity of α-amylase by aqueous extract of B. pinnatum leaves.Extract had appreciable in vitro inhibitory activity against α-amylase.The IC 50 values for the aqueous extract against α-amylase activity was determined from a dose-response curve to be 126.15± 9.76 µg/mL.polar fractions.However, within the more polar fractions, the inhibitory activity demonstrated by the ethyl acetate fraction (70.90 ± 1.23 µg/mL) was considerably higher (p < 0.05) than residual aqueous (173.85 ± 14.69 µg/mL), n-hexane (419.71 ± 26.52 µg/mL) and n-butanol (83.05 ± 3.12 µg/mL) fractions as indicated by their IC 50 values.
Acetylcholinesterase inhibitory properties of B. pinnatum leaf aqueous extract and fractions was examined and displayed in Figure 13; the result shown the ethyl acetate fraction inhibiting AChE activity in a concentration-dependent manner (20-100 μg/mL), having an IC 50 (extract concentration causing 50% inhibition) value = 66.75 μg/mL better than both the aqueous 99.05 μg/mL and n-hexane (197.42 μg/mL), n-butanol (99.41 μg/mL) and residual aqueous fractions (122.91 μg/mL).Likewise, the ability of B. pinnatum leaf aqueous extract and fractions to inhibit butyrylcholinesterase activity was also assessed, and the result is displayed in Figure 14.This revealed that the ethyl acetate fraction inhibited BChE as the concentration increases (20-100 μg/mL) having an IC 50 (extract concentration causing 50% inhibition) value = 62.97 μg/mL.

DISCUSSION
This work investigated the antioxidant activities and enzyme inhibitory properties of aqueous extract and fractions of Bryophyllum pinnatum leaf.Recently, phenolic compounds have attracted great interest for their potential use in the development of new nutraceuticals or pharmaceuticals due to their remarkable antioxidant, anti-inflammatory, anticancer or antibacterial activities.Hence, several of the possible protecting effects of polyphenols could rely upon their concentration because of high doses of some of these compounds which is also a pro-oxidant and negatively have an effect on cell growth causing toxicity. 25This might result to a multifaceted mechanism that comprises as a minimum an antioxidantpro-oxidant consequence of the polyphenolic compounds.Several present antioxidants show mutagenic and genotoxic responses in cells reflecting its oxidant activity. 25,26Flavonoids are major classes of phenolics and many studies have documented their biological and pharmacological activities. 27,28,29Though, the phenolic contents of B. pinnatum aqueous extracts and various fractions were determined respectively.Ethyl acetate fraction of B. pinnatum had higher total phenolic and flavonoid content (582.46 ± 21.25 mg GAE/g and 397.12 ± 15.46 mg RE/g) than aqueous extract and all other fractions.The reducing powers of the aqueous extracts and various fractions of B. pinnatum was evaluated supported    by its ability to scale back Fe 3+ to Fe 2+ .As displayed above, ethyl acetate fraction of B. pinnatum had higher reducing property of (3499.08 ± 45.28 mg AAE/100g) than the aqueous extract and all other fractions.Reducing properties is a defense system with two pathways obtainable to have an impact on this property are by e -transfer and hydrogen transfer. 14,30he reducing capacity of the extracts could also be a sign of its potential antioxidant activities owing to the presence of reductants.DPPH assays assess the power to scavenge radicals.The capacity to present a H atom may be a primary characteristic of antioxidants.These antioxidants present H atoms to free radicals, that converts the radicals into non-toxic species and so inhibits the propagation part of lipid oxidation. 31his study revealed that aqueous extracts (Figure 1) and various fractions from B. pinnatum leaves (Figure 7) scavenged radicals as the concentra-tion increases (20-100 µg.mL -1 ).Though, radical scavenging abilities of the ethyl acetate fractions correlates with the total phenolic contents of the leaves.Thus, the observed DPPH radical scavenging ability can be attributed to the abundant phenolic constituents in the extracts and fractions.This is consistent with previous studies. 29,32etal ion chelating ability is important since it reduces the concentration of the transition metal. 33Hence, chelating Fe 2+ , generation of hydroxyl radicals in the Fenton reaction may be attenuated and thus prevent possible damage of hydroxyl radicals to biomolecules.Accumulation of iron has been reported to lead to an increase in free radicals and development of oxidative stress. 34,35he ability of water extract and various fractions of B. pinnatum on hydrogen peroxide radical is shown in Figure 3  extracts and fractions were efficient in scavenging hydrogen peroxide radical.Though, ethyl acetate fraction of B. pinnatum leaves exhibited higher activity than the aqueous extract and all other fractions.Hydrogen peroxide may occasionally be lethal to cell and as a result give rise to hydroxyl radical within the cells though, it's not very reactive. 36Therefore, the eliminating of H 2 O 2 is extremely vital for antioxidant defense.Rice-Evans. 37reported that compounds containing phenolic could play an important in eliminating radicals.The aqueous extract (Figure 4) and fractions (Figure 10) of B. pinnatum leaves might eagerly eliminate ABTS radical specifying the presence of secondary metabolites like flavonoids, phenolic (Table 1), that validate their action.However, ethyl acetate fraction of B. pinnatum leaf recorded the utmost quantity of radical reducing ability than the aqueous extract and all other fractions.Enzyme assays have become an extremely valuable means to evaluate the potential health advantages of herbals, dietary supplements, and nutraceuticals for the development and design of functional foods or phytopharmaceuticals. 38 Besides, most usual biochemical assays involve major enzymes applicable in metabolic syndrome like diabetes (α-amylase and α-glucosidase), and Alzheimer's disease (cholinesterases). 39This study reveals that the effects of the aqueous extract and various fractions of B. pinnatum leaves were further evaluated on several selected carbohydrate hydrolysis and cholinesterase assays, respectively, and the results are shown in Figure 5, 6, 11 and 12.
The inhibition of carbohydrate metabolizing enzymes like α-amylase and α-glucosidase delays the digestion and absorption of starch and later suppresses postprandial symptom.α-amylase and α-glucosidase inhibitory activities of B. pinnatum leaf extracts and fractions are shown in (Figure 5, 6, 11, 12) been helpful as oral antidiabetic drugs for the management of high blood sugar in patients with these syndromes.Inhibitions of these enzymes interrupt macromolecule digestion and overall extent the breakdown time inflicting a discount in the degree of glucose ingestion and thus plummeting postprandial blood sugar. 40owever, aqueous extract and all the fractions of B. pinnatum leaf inhibited both α-amylase and α-glucosidase.The highest percentage inhibition of the α-amylase by the ethyl acetate fraction of B. pinnatum resulted in its low IC 50 value (62.45 ± 1.22 μg/mL) which implies that it is the most potent inhibitor of the enzyme out of the extracts and all the other fractions.This is consistent with earlier studies that antidiabetic agents derived from plants are strong inhibitors of α-glucosidase. 41,42,43This implies that ethyl acetate fractions of B. pinnatum leaf offer better pharmacological effect than the common synthetic drugs.Enzyme inhibitors delivering antioxidant roles shows potential in instances of neurodegenerative diseases, because of their defensive role against radical species. 44In spite of issues regarding absorption, metabolism and precise mechanisms of action in vivo, the screening for cholinesterase inhibitors found in plants is valid in highlighting those compounds with the most promise of biological activity. 45Besides, cholinesterase inhibitors are still the best available option for managing patients suffering from Alzheimer's disease.Hence, the aqueous extract and various fractions of B. pinnatum was active against acetylcholinesterase, whilst ethyl acetate fractions of B. pinnatum presented highest inhibitory effect (Figure 13) than the aqueous extract and all other fractions.Furthermore, for butyrylcholinesterase, ethyl acetate fractions of B. pinnatum revealed a higher inhibitory activity than aqueous extract, n-hexane, n-butanol and residual aqueous fractions (Figure 14).Ethyl acetate fractions of B. pinnatum inhibit AChE and BChE activities as the concentration increases.This is in pact with some previous studies where plant secondary metabolites revealed a considerably enhancement in reasoning and reminiscence. 46,47AChE and BChE inhibitory activity may be owing to the antioxidant properties of B. pinnatum.

Figure 5
Figure5shows the percentage inhibitory activity of α-glucosidase by aqueous extract of B. pinnatum leaves.The aqueous extract inhibited α-glucosidase in vitro.The concentration of the extract required to cause 50 % inhibition (IC 50 ) against α-glucosidase was 149.20 ± 14.44 µg/mL.Figure6shows the percentage inhibitory activity of α-amylase by aqueous extract of B. pinnatum leaves.Extract had appreciable in vitro inhibitory activity against α-amylase.The IC 50 values for the aqueous extract against α-amylase activity was determined from a dose-response curve to be 126.15± 9.76 µg/mL.Figure7shows the DPPH radical scavenging activities of the various fractions of B. pinnatum leaves.All the fractions showed an ability to quench DPPH free radicals as indicated by the concentration-dependent increase in the percentage inhibition.The ethyl acetate fraction had a consistently higher DPPH radical scavenging activity, as shown by the IC 50 (80.37± 1.54 µg/mL) than residual aqueous (98.33 ± 4.65 µg/mL), n-butanol (87.35 ± 2.11 µg/mL) and n-hexane (175.49± 15.73 µg/mL) fractions.Metal ion chelating ability of various fractions of B. pinnatum is presented in Figure8.The result revealed that the ethyl acetate fractions of B. pinnatum (79.82 ± 1.08 µg/mL) had a considerably (p<0.05)higher

Figure 7 :
Figure 7: DPPH scavenging ability of various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.

Figure 9 :
Figure 9: Hydrogen peroxide scavenging ability of various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.

Figure 8 :
Figure 8: Metal ion chelating ability of various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.

Figure 10 :
Figure 10: ABTS scavenging ability of various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.

Figure 13 :
Figure 13: Acetylcholinesterase inhibitory activities of aqueous and various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.

Figure 14 :
Figure 14: Butyrylcholinesterase inhibitory activities of aqueous and various fractions from crude aqueous extracts of Bryophyllum pinnatum leaves.
min later against a solution containing the phosphate buffer without H 2 O 2 and read at 230 nm.Hydrogen peroxide scavenging powered of both B. pinnatum extracts and fractions were calculated:Where Abs control is the absorbance of the control and Abs sample is the absorbance in the presence of the sample of B. pinnatum extracts or fractions.

Table 1 : Total phenolic, Flavonoids, Tannins, Vitamin C, reducing power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) by aqueous and various fractions of crude aqueous extract of Bryophyllum pinnatum Leaves.
This study discovered the phytochemical composition, antioxidant activity and enzyme inhibitory activities of B. pinnatum leaves.The extract and fractions of B. pinnatum scavenging radicals and had an inhibitory effect on enzymes associated to diabetes and Alzheimer's diseases in vitro.Furthermore, B. pinnatum presented a higher level of total phenolic and thus showed enhanced antioxidant and enzyme inhibitory effects.Additional studies are essential to clarify the mechanisms of in vivo antioxidant and enzyme inhibition action, bioavailability and concerned metabolic pathways.