α-Glucosidase, α-Amylase and Antioxidant Evaluations of Isolated Bioactives from Wild Strawberry

Diabetes mellitus is a metabolic disorder and is a global challenge to the current medicinal chemists and pharmacologists. This research has been designed to isolate and evaluate antidiabetic bioactives from Fragaria indica. The crude extracts, semi-purified and pure bioactives have been used in all in vitro assays. The in vitro α-glucosidase, α-amylase and DPPH free radical activities have been performed on all plant samples. The initial activities showed that ethyl acetate (Fi.EtAc) was the potent fraction in all the assays. This fraction was initially semi-purified to obtain Fi.EtAc 1–3. Among the semi-purified fractions, Fi.EtAc 2 was dominant, exhibiting potent IC50 values in all the in vitro assays. Based on the potency and availability of materials, Fi.EtAc 2 was subjected to further purification to obtain compounds 1 (2,4-dichloro-6-hydroxy-3,5-dimethoxytoluene) and 2 (2-methyl-6-(4-methylphenyl)-2-hepten-4-one). The two isolated compounds were characterized by mass and NMR analyses. The compounds 1 and 2 showed excellent inhibitions against α-glucosidase (21.45 for 1 and 15.03 for 2 μg/mL), α-amylase (17.65 and 16.56 μg/mL) and DPPH free radicals (7.62 and 14.30 μg/mL). Our study provides baseline research for the antidiabetic bioactives exploration from Fragaria indica. The bioactive compounds can be evaluated in animals-based antidiabetic activity in future.


Introduction
The use of plants or their derivatives to treat various ailments is a practice as old as human civilization [1,2]. These plants not only produce primary metabolites for their own existence but also release secondary metabolites to interact with other organisms. The secondary metabolites are usually investigated for various bioactives [3,4]. Bioactives are chemicals produced by a living being to treat various diseases or alter biological functions [5]. Over a span of decades, there have been attempts to explore the use of plants to treat various disorders, including diabetes [6,7].
Diabetes is a metabolic disorder characterized by an elevated level of blood glucose with common symptoms of polyphagia, polydipsia and polyurea [8]. There are several biochemical pathways which can be targeted for the management of diabetes [6]. The more trivial and vital enzymatic targets of diabetes are α-glucosidase, α-amylase, protein tyrosine phosphatase. The free radicals play a vital role in implications of several disorders [9][10][11]. In DM, the free radicals within the body increase due to auto-oxidation of glucose, which further complicates the situation. The free radicals damage the β cells, which are majorly responsible for the synthesis of insulin, and thus diminish the insulin synthesis [12]. The human body has the capability to combat the free radicals due to an auto-immune response [13]. However, at certain stages, when the free radicals are at uncontrolled level, the auto-immune system fails to control it [14]. At this stage of high free radicals within the body, the antioxidant treatment is necessary [15]. Reactive oxygen species and pro-inflammatory cytokines and chemokines cause the activation of JNK and NF-κB pathways that promote the development of diabetes [16]. There are 537 million adults suffering from diabetes and the number is expected to reach 783 million by 2045. The tremendous rise in diabetics confirms that it is a global challenge for policymakers and researchers to take necessary steps to overcome this challenge.
As the previous molecules are losing effectiveness, the result is certain effects, such as weight gain, heart problems, infections and weakened kidney. Despite significant additions to the list antidiabetics, researchers have to do more in search of safe, effective and efficient drug. Natural bioactives might be an effective therapeutic intervention, as studies have shown that phytochemicals are efficient agents in controlling diabetes via different mechanisms [17,18].
Fragaria indica Andrews, commonly known as wild strawberry, belongs to the family Rosaceae. It has scientific synonym Duchesnea indica (Andrews) and Potentilla indica (Andrews). It is naturalized in Africa, Europe and North America and distributed in Asian countries such as Pakistan, Kashmir, Afghanistan, Bhutan, China, India, Indonesia, Japan, Korea and Nepal [19]. The plant is known locally as the Zmaki toot, Khunmurch, and Blmngye. It is used traditionally for sore throat, tuberculosis, [20,21] nutrient, anthelmintic, and diabetic patients [22]. The plant has been explored for antioxidant, anti-inflammatory [23], and immunomodulatory potential [24,25]. Until now, other species of Fragaria such as Fragaria × ananassa and Fragaria nilgerrensis have been evaluated for their antidiabetic potentials [26,27]. However, to the best of our literature survey, we noticed that the antidiabetic potential of Fragaria indica is still unexplored. Keeping in view the global burden of diabetes and weaknesses of the available synthetic antidiabetic molecules and unexplored nature of F. indica, we plan the current work to investigate the antidiabetic and antioxidant potentials of the F. indica crude extract, semi-purified extracts and pure bioactives.

Isolation of Bioactive Compounds
In our designed study, we initially screened the crude extract and different solvent fractions of F. indica for in vitro α-glucosidase, α-amylase and DPPH activities. Based on the potency of ethyl acetate fraction (Fi.EtAc), we subjected the ethyl acetate fraction to normal gravity column chromatography with slow elution of solvent system. The solvent system was started with 100% n-hexane and was gradually increased in polarity by adding small percentages of ethyl acetate. Initially, we obtained three semi-purified phytocomponents Fi.EtAc 1, Fi.EtAc 2 and Fi.EtAc 3. The semi-purified phytochemicals were further subjected to in vitro α-glucosidase, α-amylase and DPPH activities. The semipurified components were isolated based on TLC analysis. Among the three semi-purified ethyl acetate fractions, Fi.EtAc 2 was the potent one based on its IC 50 values obtained in the in vitro assays. The Fi.EtAc 2 was further purified to obtain two bioactives 1 and 2 (as shown in Figure 1). Chemically, compound 2 is 2-methyl-6-(4-methylphenyl)-2-hepten-4-one. The molecular weight of compound 2 was confirmed as 216, and its fragmentation pattern was 216 (28%, molecular ion peak), 201 (10%), 132 (16%), 119 (46%), 83 (100%, base peak) and 55 (25%). The 1 H-NMR spectrum also showed all the proton patterns of the compound 2. The four aromatic protons appeared in two distinct doublets (each having two protons integration) at 7.12 and 7.07 chemical shift values. The observed coupling constant values (J) were 8.36 and 8.05 Hz, respectively for the two doublets. The hydrogen atom on the alkene unit of the compound 2 appeared at 6.151 as a sharp singlet. One of the methylene protons of compound 2 (-CH 2 -) gave a multiplet between chemical shift of 3.463 and 3.373. The second methylene proton (-CH 2 -) merged with methyne proton (-CH-) in the multiplet range of 3.054 to 2.963. The aromatic methyl group (Ar-CH 3 ) appeared as a singlet of three protons at 2.238. The two methyl groups attached with the alkene unit gave two distinct singlets at 2.082 and 1.994, with integration values of 3H for each one. The methyl group attached at α-position to the aromatic ring gave a doublet at 1.28 with a J value of 7.07 Hz. The 13    All values are taken as mean ± SEM (n = 3). Two-way ANOVA followed by Bonferoni test were followed. Values significantly different in comparison to standard drug, i.e., *** = p < 0.001.
Likewise, the α-amylase inhibitory potential was also in a similar pattern to that of α-glucosidase, as shown in Table 1  The α-glucosidase and α-inhibitory potentials of semi-purified ethyl acetate fractions (Fi.EtAc 1, Fi.EtAc 2 and Fi.EtAc 3) are shown in Table 3. As obvious from Table 3   All values are taken as mean ± SEM (n = 3). Two-way ANOVA followed by Bonferoni test were followed. Values significantly different in comparison to standard drug, i.e., * = p < 0.05, *** = p < 0.001 and ns: not significant.

DPPH Results
The three semi-purified ethyl acetate fractions of Fragaria indica were also found to possess strong antioxidant properties, as can be seen in Table 4. With semi-purification, the fractions showed excellent antioxidant activity profiles. The observed IC 50 values for the three fractions Fi.EtAc 1, Fi.EtAc 2 and Fi.EtAc 3 were 14.95, 20.59 and 26.25 µg/mL, respectively in DPPH free radicals scavenging activity. Based on the relative potencies and amount of each semi-purified fraction, we selected Fi.EtAc 2 for further purification to obtain the bioactive compounds.  All values are taken as mean ± SEM (n = 3). Two-way ANOVA followed by Bonferoni test were followed. Values significantly different in comparison to standard drug, i.e., ** = p < 0.01, *** = p < 0.001 and ns: not significant.

In Vitro Activities on Isolated Compounds of Fragaria indica
The two isolated compounds were characterized and were then subjected to in vitro antidiabetic activities and results are summarized in Table 5. The compound 1 (2,4-dichloro-6-hydroxy-3,5-dimethoxytoluene) and 2 (2-methyl-6-(4-methylphenyl)-2-hepten-4-one) showed excellent activities profiles exhibiting IC 50 values of 21.45 and 17.65 (α-glucosidase) and 15.03 and 16.56 µg/mL (α-amylase), respectively. Similarly, of the two compounds, specifically, the phenolic type of derivative (1) was the more potent antioxidant. The observed IC 50 values for compounds 1 and 2 were 7.62 and 14.30 µg/mL, respectively against the DPPH free radicals. The standard drug IC 50 under the same set of condition was 4.98 µg/mL.

Discussion
Researchers, specifically the medicinal chemists and pharmacologists are constantly searching for new drug molecules [28][29][30]. The organic chemists design and explore new methods for the synthesis of valuable molecules which can be important drug or natural products [31,32]. On the other hand, the medicinal chemists are in constant evaluation of medicinal compounds for various vital targets [33][34][35]. The pharmacological researchers use various in vitro and in vivo methods to confirm the possible use of a compound for a specific disease [36]. One of the major tools is the computational approach, which allows researchers to obtain the binding energies for a specific biological target [37]. The medicinal values of compounds can be explored from natural and synthetic origins [38,39]. Among the major health issues, diabetes is a global challenge to the researchers. Various natural and synthetic origins have been employed for the management of diabetes [40]. However, due to the emerging increasing number of diabetes patients, more natural sources can be explored to treat diabetes. Poly-and oligosaccharides are converted into monosaccharides by the action of intestinal α-glucosidase and α-amylase, and the activity significantly contributes to postprandial hyperglycemia. Synthetic agents such as acarbose can inhibit these enzymes, but the side effects, such as diarrhea, flatulence, and stomach pain, make them unsuitable [41]. The present study was designed to develop antidiabetics from natural products based on this strategy with lesser side effects [42].
The ethyl acetate fraction of F. indica showed maximum inhibition of α-glucosidase, α-amylase enzyme and DPPH free radical among the tested fractions and crude extract. The inhibitory activity was enhanced with semi-purification of the ethyl acetate fraction. The isolated compounds showed equivalent potent activities to the standard used.
The wild-type strawberry, i.e., F. indica herb was investigated for the first time for in vitro antidiabetic and antioxidant activities. However, other species, especially fruits, are extensively investigated for such activities. The enzyme α-amylase and α-glucosidase activity were inhibited by ellagic acid or derivatives in aqueous fruit extract of Fragaria × ananassa [26]. Similarly, another specie F. vesca leaf extract was reported to inhibit αglucosidase and α-amylase enzyme activity [41]. Flavonoids in n-butanol extract of F. nilgerrensis Schlecht produced an antidiabetic effect [27].
The antioxidant properties of strawberries have been well documented, both in vitro and in vivo [48][49][50]. A study on aqueous extract of Fragaria vesca L. collected in Bulgaria showed strong ABTS inhibitory activity due to more polyphenols than those with less ABTS inhibitory potentials [51].
Strawberries and strawberry-based phytochemicals are potential dietary sources for managing hyperglycemia [52]. The leaves of the Fragaria × ananassa Duch. cultivars have also been reported for antihyperglycemic and antioxidant potentials [53].
The beneficial effects of the different strawberry extracts on blood glucose levels are popularizing its role as functional food [54,55]. The role was further supported by a clinical trial where a diet supplemented with strawberry fruit extract lowered glycohemoglobin (HbA1c) levels after six weeks of treatment [56].
Our experiments revealed that polyphenolic compounds of ethyl acetate fractions, e.g., compounds 1 and 2, are potential major components possessing anti-diabetic and antioxidant potentials; however, further analytical and structure-activity studies are required to identify and synthesize more active components in the field.

Collection of Medicinal Plant and Extraction
The plant was collected from Laram Qilla Talash (Latitude 45.990530, Longitude 12.673570), District Dir Lower of Khyber Pakhtunkhwa (KPK) Pakistan in August 2020 and was identified as Fragaria indica Andrews by Nasrullah, Plant Taxonomist at the department of Botany University of Malakand, Chakdara, Pakistan. A sample specimen was deposited at the herbarium of the same department, voucher no. H.UOM.BG.551. The extraction was carried out as per our previous mentioned procedure [57]. The crude extract was subjected to different solvents fractions based on the polarity basis as per our previously reported procedure [58].

Phytochemistry and Bioactives Isolation
In our initial in vitro studies, we observed that among all the fractions, ethyl acetate fraction (Fi.EtAc) was the most prominent. Based on this, the ethyl acetate fraction was subjected to gravity column. The column elution was started with 100% of pure n-hexane as non-polar component. The polarity of solvent was gradually increased with the addition of ethyl acetate as polar modifier solvent. After the column chromatography, we were able to collect three different ethyl acetate fractions (Fi.EtAc 1-3) as visualized on precoated TLC plate under UV lamp. The three semi-purified ethyl acetate fractions were also subjected to all in vitro assays. Among the semi-purified ethyl acetate fractions, Fi.EtAc 2 was observed with potent IC 50 values in our in vitro experiments. Based on the potency, Fi.EtAc 2 was further subjected to purification to obtain the bioactive compounds. At the end of purification process, we were able to obtain two bioactive compounds (1 and 2). The isolated compounds were characterized by their masses and also their structures were confirmed by proton NMR [6].

In Vitro α-Glucosidase Inhibition
α-Glucosidase inhibitory assay on F. indica extracts, solvent fractions and isolated compounds were performed according to the reported procedure [59] using acarbose as standard drug. The α-glucosidase solution (120 µL) of 0.50 U/mL in 0.10 M phosphate buffer (pH 6.90) was prepared and p-nitrophenyl-α-D-glucopyranoside (5 mM) was added as substrate solution. Different concentrations ranging from 62.50 µg/mL to 1000 µg/mL of the crude extracts, semi-purified fractions and isolated compounds of F. indica were prepared accordingly. The plant's samples (extracts, semi-purified and purified compounds) were mixed with the solutions of enzyme as per the experiment and were incubated at 37 • C.
After initial incubation for 15 min, 20 µL p-nitrophenyl-α-D-glucopyranoside solution was mixed and was again incubated in the same way. Finally, 80 µL solution of sod. carbonate (0.20 M) was added to finish up the reaction. The solution containing all the substrates except α-glucosidase served as a blank. The absorbances of experiments were measured at 405 nm. The experiments were repeated 3 times and the calculations were made as per the standard protocols.

In Vitro α-Amylase Inhibition
The α-amylase inhibitory activity was carried out according to the established protocols for F. indica extracts and isolated compounds [60] The amylase solution was prepared in phosphate buffer. The solutions of the F. indica and isolated compounds (62.50 to 1000 µg/mL) were added to the amylase solution and allowed to react. After reaction completion, starch solution was added and incubated as per the standard method. Afterwards, a solution of dinitro-salicylic acid was added to both test and control groups. The final solutions were heated for 3 min in boiling water and the absorbances were measured at 656 nm. The experiments were performed in triplicates and percent inhibitions were calculated.

DPPH Free Radicals Scavenging Assay
The antioxidant activity was carried out for F. indica extracts, semi-purified ethyl acetate fractions and isolated compounds using DPPH free radicals. DPPH solution was prepared (24 mg/100 mL of methanol). Stock solutions (1 mg/mL) of F. indica extracts and isolated compounds were prepared in methanol and then diluted to 1000, 500, 250, 125, 62.5 µg/mL. Sample and DPPH solutions were mixed in a ratio of 1:1 and were incubated at 23 • C for 30 min. Finally, absorbance was measured at 517 nm using UV spectrophotometer (Thermo electron corporation, Waltham, MA, USA). Ascorbic acid was used as positive control. Percent radical scavenging activity was measured using the reported equations [61].

Estimation of IC 50 Values
The half inhibitory concentration (IC 50 ) was calculated using Microsoft Excel program as per our previously reported method [62].

Statistical Data Analysis
The results in assays were expressed as mean ± S.E.M. GraphPad prism (GraphPad Software, San Diego, CA, USA) was used for one-way ANOVA followed by Dunnett's multiple comparison test with positive control and test groups. The p values less than 0.05 were considered as statistically significant. The level of significance was expressed as * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

Conclusions
It can be concluded from our current research results that Fragaria indica is a rich source of bioactives. Herein, we have explored the Fragaria indica for its antidiabetic potential. We initially confirmed from crude extracts' in vitro assays that ethyl acetate fraction of the wild strawberry was potent among all. Then, we semi-purified ethyl acetate fractions Fi.EtAc 1 to 3. The Fi.EtAc 2 was the fraction with dominant activities. We were able to isolate two compounds, i.e., 1 (2,4-dichloro-6-hydroxy-3,5-dimethoxytoluene) and 2 (2-methyl-6-(4-methylphenyl)-2-hepten-4-one). The two compounds showed overwhelming activity profile against α-glucosidase, α-amylase and DPPH free radicals. The two compounds can be further evaluated for antidiabetic targets using in vivo models to obtain more fruitful results.