INTRODUCTION:

Globally, medicinal plants have been used as a source of medicine, and 80-85% of people use the extracts or the active ingredients of these plants as their primary source of traditional medicine¹. Several active ingredients have been extracted from medicinal plants for use as medicines, lead compounds, or pharmacological agents.

Diabetes mellitus, one of the major public health problems worldwide, is a metabolic disorder of multiple etiologies distinguished by a failure of glucose homeostasis with disturbances of carbohydrate, fat, and protein metabolism as a result of defects in insulin secretion and insulin action². Chronic hyperglycemia promotes oxidative stress in all tissues because glucose generates reactive oxygen species in large numbers.

Acarbose, Miglitol, and Voglibose, which are the primary components of medications used to treat diabetes, work by inhibiting α -glucosidase and α-amylase³. Due to the side effects of these synthetic drugs different plant families have been used traditionally throughout the world to manage diabetes since they are less expensive and hazardous than the drugs acarbose and miglitol that are currently used to treat it. Numerous medicinal plants are used to treat diabetes in the traditional medical system⁴. Some of these include Allium sativum, Aloe vera, Coccinia indica, Eugenia jambolana, Ficus bengalensis, Momordica charantia, and Ocimum sanctum. Others have been scientifically tested for their anti-diabetic efficacy. The phenolic compounds Physcion, C-glycosylated anthrones, 2-hydroxy-3-methylanthraquinone, and anthroquinones are produced by medicinal plants and show anti-diabetic properties⁵.

ROS also creates oxidative stress in cells, which can contribute to infectious and inflammatory diseases. Inflammation usually occurs when infectious microorganisms such as bacteria, viruses, or fungi when entering the body, live in specific tissues, or circulate in the circulation⁶. Numerous phagocytic cells, including mononuclear cells and polymorphonuclear leukocytes, which are essential to the host defense system, produce an excessive amount of ROS⁷. These excessively produced ROS impair cellular processes, leading to cellular and tissue damage, which exacerbates the inflammatory state in addition to their defensive functions. Analgesics, nonsteroidal anti-inflammatory medicines (NSAIDs), and steroids (corticosteroids) are prescribed medications that act to reduce pain and inflammation⁸. Over 30 million people use non-steroidal anti-inflammatory medicines daily for inflammation and other ailments, making it one of the most commonly prescribed drug classes in the world. Most NSAIDs, such as aspirin (salicylate derivatives), indomethacin (carboxylic and heterocyclic acid derivatives), and propionic acid derivatives (ibuprofen, ketoprofen, and flurbiprofen), contain carboxylic acids (diclofenac)⁹. Traditional Ayurvedic medicine has used a variety of herbs to manage and cure a variety of inflammatory disorders and wound healing procedures¹⁰. Consequently, scientists are continually interested in finding novel natural inhibitors in plants due to studies indicating possible adverse effects that may be related to the consumption of synthetic drugs¹¹.

Heterospathe elata belongs to the Arecaceae family. The Arecaceae family has about 2600 species in 181 genera¹². These species are primarily found in tropical and subtropical temperatures as well as arid regions and desert environments¹³. Arborescent palm trees are also occasionally found in this family. The biological effects of Heterospathe elata fruit have not been verified by scientists. According to pharmacological research, plants of the Arecaceae family, such as Cocus nucifera and Borassus flabellifer, have potent anti-inflammatory and anti-diabetic properties^14-15. The goal of the current investigation was to explore Antidiabetic properties by alpha-amylase and alpha–glucosidase inhibitory assay and anti-inflammatory activity by bovine serum protein denaturation assay and HRBC membrane stabilization of Heterospathe elata fruit.

MATERIAL AND METHODS:

Collection of the plant:

Heterospathe elata plants were collected from the Northern Indian state of Uttar Pradesh in July 2021 and were identified by an expert taxonomist at the Department of Botany, Banaras Hindu University. A voucher specimen (1/7 A, 22-7-21) was deposited to the herbarium in the Department of Botany, University of B.H.U.

Chemicals and Instruments:

Nitrophenyl-α-D-glucopyranoside (SRL Pvt., Ltd), Tris buffer (Merck), α-amylase ex porcine pancreas (SRL Pvt., Ltd), Dimethyl superoxide (DMSO) (Merck), 3,5-dinitro salicylic acid (DNSA) (SRL Pvt., Ltd), α-glucosidase for biochemistry ex microorganism (SRL Pvt Ltd); and acarbose, Diclofenac sodium (Cipla Ltd, Bangalore), sodium carbonate (CDH) were purchased. All other chemicals, solvents, and reagents used were of analytical grade. UV-Visible spectrophotometer (Systronics 118) was used for the analysis.

Preparation of Extract:

The fruit of the plant was cleaned with running tap water followed by distilled water. The fruit sample was air-dried. After drying the dried materials were ground into a coarse powder with the help of a grinding machine (Lab mill grinder Arthur H. Thomas, Star Scientific Instruments, Delhi), and the powdered materials were stored at room temperature for future use. Extraction was carried out by soxhlet apparatus, successively using solvents in increasing order of polarity Isopropyl alcohol, Hydroalcohol, and water. After extraction and filter the extract and concentrated under reduced pressure by using a rotatory evaporator (Perfit, India), and the extract was stored for further investigation. For each solvent, the extraction process was done in triplicate.

Figure 1: Detailed extraction process of Heterospathe elata fruit

Evaluation of in vitro antidiabetic activity:

Alpha-amylase inhibitory assay:

The enzyme inhibition procedure of α-amylase was according to the method of Kazeem et al with some modifications¹⁶. The amount of reducing group produced when isolated pancreatic-amylase hydrolyzes soluble starch allows for the measurement of α–amylase activity. 3,5-dinitrosalicylic acid is reduced to nitroaminosalicylic acid, which causes a color shift and a change in the absorbance at 540nm. α - amylase inhibitor that prevents starch hydrolysis reduces absorbance at 540nm in comparison to the control. As a reference drug for α- amylase Acarbose was used. The half maximum inhibitory concentration was also used to express the percentage (%) of inhibition for α- amylase (IC₅₀). The following is the formula for % inhibition:

Absorbance of control – Absorbance of extract

% of Inhibition = --------------------------------------- X100

Absorbance of control

Alpha-glucosidase inhibitory assay:

The enzyme inhibition procedure of α-glucosidase was according to the method of Kim et al with slight modification¹⁷. The enzyme α-glucosidase breaks down the substrate p-nitrophenyl-α-D-glucopyranoside (p-NPG) (SRL Pvt. Ltd.) into p-nitrophenol and D-glucose, respectively, when it is incubated with the substrate. As a reference medication for the assay for α-glucosidase inhibition, acarbose (AC) was utilized. At 410nm, the emitted p-nitrophenol was measured. Each experiment was run in triplicates with a control set up inparallel without test samples. The half-maximal inhibitory concentration was also used to express the percentage of inhibition (%) for all enzymes other than α-glucosidase (IC₅₀).

In vitro anti-inflammatory activity:

Inhibition by bovine serum denaturation method:

The ability of plant fruit extract to protein denaturation was determined according to the method described by Chandra et al with some modifications¹⁸. The reaction mixture consists of 2.8mL of phosphate-buffered saline (pH 6.4), 0.2mL of bovine albumin, and 2 mL of various extracts at variable concentrations. The reaction mixtures were then incubated in the BOD incubator for 15minutes at ambient temperature before being heated for 5minutes at 72^οC. Their absorbance at 660nm (Systronic 118, UV-Vis) was measured using a blank after cooling. To determine absorbance, sodium diclofenac was utilizedas a reference medication and handled similarly to plant extracts. The following equation wasused to determine the %inhibition of protein denaturation:

Absorbance of control – Absorbance of extract

% of Inhibition = --------------------------------------- X100

Absorbance of control

HRBC Membrane stabilization method:

The in vitro HRBC membrane stabilization method was used to evaluate the anti-inflammatory efficacy of several extracts from the fruit of Heterospathe elata¹⁹. The blood was centrifuged with isosaline after being combined with an equal amount of Alsever's solution. Equal volumes of the test substance at the concentrations of 50, 100, 250, 500, and 700µg/mL were added to 1mL of HRBC solution. All of the assay mixtures under went a 30-minute incubation period and a 37°C centrifugation. A UV-Visible spectrophotometer set to 560nm was used to determine the amount of hemoglobin in the supernatant solution.

Statistical analyses:

All the experiments were done in triplicates and the results were expressed as Mean±SD. The data were statistically analyzed using one way ANOVA followed by Duncan’s test. Mean values were considered statistically significant when p>0.05.

RESULT:

Antidiabetic Activity:

The in–vitro α-amylase inhibition assay of Heterospathe elata fruit extract compared with standard drug acarbose. Fig 2 shows the alpha-amylase % inhibition of fruit extracts and standard acarbose, hydroalcohol extract shows the highest % alpha-amylase of 38.19% - 94.34% on varying concentrations of 50-1500μg/ml. A lower IC₅₀ value corresponds to greater potency and better therapeutic efficacy. The IC₅₀ value of hydro alcohol extract is close to acarbose and thus can be regarded as an excellent alpha-amylase inhibitor (Table 1). Standard acarbose showed an alpha-amylase inhibition percentage of 35.18% - 91.55% on varying concentrations of 50-1500μg/ml with an IC₅₀ value of 48.82μg/ml greater potency and better therapeutic efficacy.

Table 1: IC₅₀value of α- amylase inhibitory assay of Heterospathe elata fruit

S. No	Plant Extract	IC₅₀(µg/ml)
1	Isopropyl alcohol	761.6 ±0.02
2	Hydroalcohol	75.71±0.15
3	Water	468.71±0.23
4	Acarbose	48.82±0.34

Figure 2: Alpha-amylase inhibition of Heterospathe elata fruit extracts

The in vitro α-glucosidase % inhibitory activity of Heterospathe elata fruit extracts compared with standard drug acarbose. Fig 3 represents the α-glucosidase inhibition on varying concentrations of each fruit extract. The hydroalcoholic extract shows the highest alpha-glucosidase inhibitory activity (33.43% - 96.93%) on varying concentrations of 50-1500μg/ml. Table 2 represents the IC₅₀ value of each extract as well as standard acarbose. Hydroalcoholic extract show the highest alpha -glucosidase inhibition assay (IC₅₀ = 78.15±0.17μg/ml) it is close to standard drug acarbose

(IC₅₀= 48.82μg/ml).

Table 2: IC₅₀ for α- glucosidase inhibitory assay of Heterospathe elata fruit

S. No	Plant Extract	IC₅₀(µg/ml)
1	Isopropyl alcohol	720.78±0.56
2	Hydroalcohol	78.15±0.17
3	Water	344±0.09
4	Acarbose	48.82±0.22

Figure 3: Alpha-glucosidase inhibition of Heterospathe elata fruit extracts

Anti-inflammatory Activity:

In- vitro anti-inflammatory activity evaluated by Bovine serum albumin assay of Heterospathe elata fruit extract compared with Sodium diclofenac standard drug is illustrated. Fig 4 shows the percentage protein denaturation inhibition on the varying concentration of each extract, hydroalcohol shows the highest % inhibition of protein denaturation at 33.56% - 94.61% on varying concentrations of 100-1500μg/ml. Table 3, show the IC₅₀ value of each fruit extract as well as standard sodium diclofenac. The hydroalcoholic extract shows the highest protein denaturation assay (IC₅₀ = 105.27 μg/ml ). The IC₅₀ value of hydro alcohol extract is close to sodium diclofenac and thus can be regarded as an excellent protein denaturation. Standard sodium diclofenac showed % inhibition of protein denaturation of (36.78% - 96.88%) on varying concentrations of 50-1500μg/ml with IC₅₀ value 95.76μg/ml greater potency and better therapeutic efficacy. The anti-inflammatory activity of Heterospathe elata fruit is in the following order Hydroalcohol> water > Isopropyl alcohol extract.

Table 3: IC₅₀value for Bovine serum denaturation assay of Heterospathe elata fruit

S. No	Plant Extract	IC₅₀(µg/ml)
1	Isopropyl alcohol	762±0.02
2	Hydroalcohol	105.27 ±0.34
3	Water	179.72±0.19
4	Sodium diclofenac	95.76±0.43

Figure 4: Anti-inflammatory assay of Heterospathe elata by Protein denaturation assay

In- vitro anti-inflammatory activity evaluated by HRBC membrane stabilization of Heterospathe elata fruit extract compared with aspirin standard drug is illustrated. Fig 5 show the %inhibition on varying concentration of each extract, hydroalcohol show the highest % inhibition on varying concentration of 100-1500μg/ml. Table 2, show the IC₅₀ value of each fruit extract and as well as standard aspirin. The IC₅₀ value of hydro alcohol extract is close to aspirin and thus can be regarded as an excellent membrane stabilizer. Standard aspirin showed % inhibition (20.14% - 97.36%) on varying concentrations of 50-1500 μg/ml with an IC₅₀ value of 71.32μg/ml greater potency and better therapeutic efficacy. The anti-inflammatory activity of Heterospathe elata fruit is in the following order Hydroalcohol> water > Isopropyl alcohol extract.

Table 4: IC₅₀ value for HRBC membrane stabilization assay of Heterospathe elata fruit

S. No	Plant Extract	IC₅₀(µg/ml)
1	Isopropyl alcohol	779.78 ±0.76
2	Hydroalcohol	200.52±0.04
3	Water	381.62±0.15
4	Aspirin	71.32±0.25

Figure 5: Anti-inflammatory assay of Heterospathe elata by HRBC membrane stabilization assay

DISCUSSION:

Research collaborations between the manufacturing sector and academics have revolutionized the extraction of natural products using biocompatible solvents²⁰. It is a brand-new innovation sector that will continue. A bio-viable substitute for traditional solvents is made possible by the efficient extraction of different phytochemicals using "green" solvents²¹. Chemical operations are economically and environmentally viable thanks to the modest non-toxicity of this solvents. Herbal therapy is quite effective in the treatment of many different diseases²².

Numerous new bioactive phytoconstituents with hypoglycemic and anti-hyperglycemic properties that demonstrate some anti-diabetic activity are derived from plants²³. Alkaloids, phenolics, flavonoids, and triterpenoids make up this phytoconstituents²⁴. The current study examined the fruit of Heterospathe elata in vitro anti-inflammatory and anti-diabetic properties. Antidiabetic properties were evaluated by alpha-amylase and alpha-glucosidase inhibition assay. Insulin is released by pancreatic ß-cells, preventing the absorption of glucose²⁵. These phytochemicals are known to heal the damaged beta cells and stop oxidative stress on beta cells in addition to their effects on reducing blood glucose levels²⁶. According to our research, the hydro alcohol extract exhibited the most significant inhibition, whereas the effect of the isopropyl extract was the least significant. Polar chemicals are most likely to blame for the hydro alcohol extract inhibiting effects on alpha-amylase and alpha glucosidase. The antidiabetic activity of Heterospathe elata fruit is in the following order Hydroalcohol > water > Isopropyl alcohol extract.

In this study, the bovine serum protein denaturation bioassay was used to evaluate the anti-inflammatory properties of the fruit extract from Heterospathe elata. The underlying factor in inflammatory diseases is tissue protein denaturation²⁷. Due to the denaturation of protein antibodies created against some inflammatory diseases, it is necessary to control the inflammatory response by preventing the release of lysosomal components of activated neutrophils, such as bacterial proteases, which cause auxiliary tissue inflammation and extracellular release²⁸. Consequently, it would be useful for the development of anti-inflammatory medications if characteristics that limit protein denaturation, such as solubility, were considered²⁹. Previously, by many scientists the effect of different plant extracts on protein denaturation has been evaluated for example, Semecarpus anacardium bark on bovine albumin, an ethanolic extract of Wedelia trilobate bovine albumin Albucas erosion egg albumin, etc^30-31. Our research revealed that hydroalcohol extracts have high anti-inflammatory activity, due to the presence of high content of flavonoid in hydroalcohol extract. It follows logically that the anti-inflammatory properties of the standard drug may be a result of the extracts anti-inflammatory activity. Therefore, it can be inferred from this study, Heterospathe elata fruit showed a substantial in vitro anti-inflammatory activity against protein denaturation.

CONCLUSION:

In this work, green solvents like isopropyl alcohol, hydro alcohol, and water were used for the extraction of Heterospathe elata fruit. The present study revealed that the hydro alcohol extract of Heterospathe elata fruit could be of greater importance as a therapeutic agent in preventing diabetes and inflammation related disorders. Further studies are currently underway to assess the in vivo biological activities and to identify the active component responsible for their antidiabetic and anti-inflammatory properties.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this study.

ACKNOWLEDGMENTS:

The authors are grateful to Department of Chemistry, KGC, Gurukula Kangri (Deemed to be University), Haridwar for providing all the necessary facilities.

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Received on 15.09.2022 Modified on 11.04.2023

Research J. Pharm. and Tech 2024; 17(1):163-168.

DOI: 10.52711/0974-360X.2024.00026