{"id":57649,"date":"2024-06-25T11:00:30","date_gmt":"2024-06-25T11:00:30","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=57649"},"modified":"2024-07-03T17:25:38","modified_gmt":"2024-07-03T17:25:38","slug":"pharmacological-characterization-of-nigella-sativa-extract-and-the-compound-beta-caryophyllene","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no2\/pharmacological-characterization-of-nigella-sativa-extract-and-the-compound-beta-caryophyllene\/","title":{"rendered":"Pharmacological Characterization of Nigella sativa Extract and the Compound Beta-caryophyllene"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Medicinal plants have various forms like\nextracts, oils, and compounds that are identified to have anti-AD,\nanti-diabetic, and anti-inflammatory properties and have also undergone many\nclinical and pre-clinical studies. Dementia is defined as an acquired deterioration in cognitive\nabilities that impairs the successful performance of activities of daily living. The memory impairment in Alzheimer\u2019s disease\n(AD) patients is due to the loss of cholinergic neurons, while those in non-AD\ndementia are due to the loss of serotonergic and glutaminergic neurons. While\nin the former, memory impairment is primary, in the latter, behavioral symptoms\nare primary, leaving memory relatively spared. The authors\nsuggestions have led several researchers to conclude that the presence of\ncertain types of alkaloids, saponins, and glucosides, as well as antioxidant\nactivity, possess a neuroprotective effect against A\u03b2 production, causing AD,\nwhich tends to be high. As medicinal plants are rich biosources of drugs from\nplant sources, they have been widely used in many research studies and\nnutraceuticals, the food industry for preparing food supplements, the drug\nmanufacturing industry, and also as Siddha and Ayurvedic medicines. The most\ncommon and native treatment used in India is Ayurvedic medicine. The treatment\nand cure for diseases are through medicinal plants, which are evaluated and\ndesigned for potential cure 1<\/sup>.<\/p>\n\n\n\n

For many health-related diseases and\ndisorders various phytotherapies are used, which are obtained from herbal medicines.\nIt causes a major change in the outlook for treatment around the world.\nAccording to research, herbal plants are considered to be the best remedy and\nhave holistic therapy, which consolidates the physiological status of the\npatients 2 <\/sup>. The efficient and effective herbal medicines contain\nseveral compounds that actively interact with the in vivo <\/em>environment\nand change the physiological status of the body through certain alterations in\nthe biochemical processes without any side effects 3<\/sup>. Efficient and effective herbal medicines contain several\ncompounds that actively interact with the in-vivo\n<\/em>environment and change the physiological status of the body through certain\nalterations in the biochemical processes without any side effect. 3<\/sup><\/p>\n\n\n\n

Nigella sativa<\/em>, a well-known spice plant (black cumin) of the Ranunculacea\nfamily, exhibited many significant therapeutic properties. The active compounds\nof Nigella sativa<\/em> have made it to be used as a medicinal plant for thousands\nof years and also as medicinal ailments 4<\/sup>. The drugs that are formulated are from the plant\u2019s stem, bark,\nflowers, leaves, roots, seeds, and fruits. The phytochemical constituents are\nthe most important sources for designing a drug 5,6<\/sup>. Various\nessential oils and extracts, which are the richest source of phytochemicals and\ncontain tannins, carbohydrates, alkaloids, terpenoids, phenolics, flavonoids,\nand steroids 7<\/sup>. Many studies are correlated with herbal extracts,\nwhich are a good source of phytochemical constituents, have anti-diabetic and\nanti-inflammatory activities, and have good potential for enzymatic assays. The generation of reactive oxygen species causes adverse\neffects in aerobic organisms, but the metabolism of oxygen has more beneficial\neffects. The macromolecules mostly undergo oxidative reactions that are\nmediated by reactive oxygen species. Biomedical research mainly focuses on the\nadverse effects of reactive oxygen species, which act upon the biological\nsystem 8<\/sup>. Based on the experimental studies and clinical studies, pharmacological\nactivity occurs due to the antioxidant activity of the extract. Because it has\nthe ability to scavenge free radicals and inhibit lipid peroxidation 9<\/sup>.\nMany studies and researches have concluded that the seed extract of Nigella sativa <\/em>and its derivative products were used as a treatment for\nmany diseases, like liver diseases, rheumatism, as well as in the treatment of\ninflammatory disorders and their relevant consequences 10<\/sup>. Further,\nthe constituents of the extract are found to be effective, and this is evident\nas it decreases the nephrotoxicity induced by cisplatin in rodents and possesses\nhigh anti-tumor activity 11<\/sup>. Most of the therapeutic properties are\ndue to the presence of some phenolic compounds in the seed, especially thymoquinone,\nbeta-caryophyllene, etc which are known to be the major bioactive component. The present study was carried out using Nigella sativa<\/em>\nextract and the compound beta-caryophyllene to phytochemically analyze the\neffects of the extract and the compound on Alzheimer\u2019s disease.<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Reagents<\/strong><\/p>\n\n\n\n

Amyloid\npeptide, Thymoquinone, beta-caryophyllene, 1,1,1,3,3,3-hexafluoro-2propanaol\n(HFIP) was purchased from Sigma Aldrich; 1, 1-diphenyl-2-picrylhydrazyl (DPPH),\nAscorbic acid, Dimethyl Sulfoxide (DMSO), Triton X 100, and p-Nitrophenyl –\n\u03b1-D- glucopyranoside were obtained from AR Teck Pvt. Ltd., India. All the\nchemicals used for the experiments are of analytical grade. <\/p>\n\n\n\n

Anti-oxidant\nactivity of ESENS and compound beta-caryophyllene<\/strong><\/p>\n\n\n\n

The\nantioxidant activity of compounds such as 2,2 -diphenyl-1-picrylhydrazyl\n(DPPH), hydrogen peroxide, and nitric oxide was studied. The DPPH radical\nscavenging capacity was measured using the Brand-William method 12<\/sup>. Similarly,\nhydrogen peroxide and nitric oxide were measured using Ruch and Marcocci\nmethods 13,14<\/sup>.<\/p>\n\n\n\n

2,2\nDiphenyl-1-Picrylhydrazyl (DPPH) radical scavenging assay of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

Ethanolic\nseed extract of Nigella sativa <\/em>(ESENS) and compound beta-caryophyllene\nobtained through HR-LCMS were pre-dissolved using DMSO and used for the study.\nThe compounds and the extract in various concentrations were taken and added to\n100 \u03bcl of 0.1 mM DPPH, which was freshly prepared using ethanol. DPPH alone\nserves as a blank, and ascorbic acid is the standard. The solution mixture was\nfurther incubated for 30 minutes. The incubated mixture was studied using an\nabsorbance of 517 nm using a UV-Vis spectrophotometer. The triplicates obtained\nwere used to calculate the percentage of radicals that were being scavenged.<\/p>\n\n\n\n

Hydrogen\nperoxide scavenging assay<\/strong><\/p>\n\n\n\n

Based\non the Ruch and Marcocci method, using phosphate buffer, 40 mM of hydrogen\nperoxide was freshly prepared, and the pH was adjusted to 7.4. Various\nconcentrations of the ESENS and the compound beta-caryophyllene were added to\nthe freshly prepared hydrogen peroxide, which was further incubated for 30\nminutes at RT. Phosphate buffer alone serves as a blank, and ascorbic acid is\nused as a standard. After the incubation period, the samples were measured at\n560 nm using UV-Vis spectrophotometry. Triplicates obtained were used to\ncalculate the percentage of inhibition that the compounds beta-caryophyllene\nand ESENS had undergone. <\/p>\n\n\n\n

Nitric oxide radical scavenging assay <\/strong><\/p>\n\n\n\n

Based\non modified protocol 15<\/sup>, the nitric oxide radical scavenging assay\nwas performed for the various concentrations of ESENS and the compound\nbeta-caryophyllene. Equal amounts of Griess reagent were mixed with\nsulphanilamide (1%), which was prepared from 2.5% phosphoric acid, and\nnapthylethylene diamine dihydrochloride (0.1%) in 2.5% phosphoric acid. To\ndifferent concentrations of Ethanolic Seed Extract of Nigella sativa <\/em>(ESENS)\nand compound beta-caryophyllene, 0.5 ml of 10 mM sodium nitroprusside, which\nwas prepared using phosphate buffered saline, was added. Further, the tubes\ncontaining the reaction mixture were incubated at 25\u00b0C for 180 minutes, followed\nby the addition of an equal amount of freshly prepared Griess reagent. For\ncontrol, the reaction mixture was prepared with buffered saline without ESENS\nand the compound beta-caryophyllene. The samples were read at 546 nm using\nUV-Vis Spectrophotometers. Ascorbic acid is used as a standard. The percentage\nof inhibition was observed and recorded to analyze the percentage of nitrite\nscavenging activity of the extract, compound, and standards.<\/p>\n\n\n\n

Anti-invitro<\/em>\nstudies of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n\n\n\n

Increasing levels of proinflammatory cytokines cause metabolic disorders that lead to cell death. The ESENS and the compound beta-caryophyllene were studied to find out how they stop the proinflammatory cytokines that cause metabolic disorders.16,17<\/sup>   <\/strong><\/p>\n\n\n\n

HRBC membrane stabilization assay of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

To the tube\ncontaining EDTA, 100 \u03bcl of blood sample and various concentrations of ESENS and\nthe compound beta-caryophyllene were added. Triton x 100 serves as a positive\ncontrol, while a blood sample alone serves as a negative control. The tube\ncontaining the reaction mixture was incubated at 37\u00b0C for 30 minutes. Followed\nby the centrifugation of the sample at 5000 rpm for 15 minutes. The supernatant\nis collected in a separate tube for analysis and stabilization using UV-Vis\nSpectrophotometer at 517 nm. <\/p>\n\n\n\n

Cytotoxicity assay of ESENS and compound the beta-caryophyllene<\/strong><\/p>\n\n\n\n

The isolated lymphocyte cells are cultured in\nhumidified 5% (v\/v) CO2 \/ air at 37\u00b0C in Dulbecco\u2019s Modified Eagle Medium\n(DMEM), which is supplemented with 10% fetal bovine serum and 100 U\/ml of\npenicillin. In 96-well plates, 5×104<\/sup> cells\/ml were cultured.\nSimultaneously, amyloid beta fibrils were prepared by incubating the amyloid\nmonomer at room temperature. The pre-formed amyloid beta fibrils with ESENS and\ncompound beta-caryophyllene and without ESENS and compound are diluted in\nfreshly prepared DMEM medium and added to a microtiter well plate; the wells\nfinal concentration was made to 2\u03bcm ol\/L. The same volume of medium is added to\nthe control well. The plates were then incubated at 37\u00b0C for 48 hours. Cell\nviability was determined by using the\n3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) toxicity\nassay. The MTT was prepared at a concentration of 5mg\/ml; added to each well,\nand incubated further for 3 hours at 37\u00b0C. In the medium that was removed, DMSO\nwas added to each well. The samples in the wells were mixed well, and the\nsamples were read using a microplate reader at 490 nm. 18,19<\/sup>         <\/p>\n\n\n\n

Enzyme linked immune-sorbent assays of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

Interleukin \u2013 1 \u2013 beta were analyzed using lymphocyte\ncell culture supernatant and studied using commercially available ELISA kit\nprotocols. The lymphocytes were collected, and 100 \u00b5l of the sample was added\nto each well of the 96-well microtiter plates along with an equal volume of\nDMEM medium. The samples were further incubated for 2 hours at 37\u00b0C. The\nincubated samples were again aspirated and washed 3 times. Further, 100 \u00b5l of\ndetection reagent B was added and further incubated for 30 minutes at 37\u00b0C. The\nsamples are washed 5 times. To achieve this, 90 \u00b5l of substrate solution was\nmixed and incubated for 10\u201320 minutes at 37 \u00b0C. To terminate the reaction, 50\n\u00b5l of the stop solution was mixed, and the absorbance was measured at 450 nm\nimmediately after mixing the stop solution.<\/p>\n\n\n\n

Anti-diabetic activity of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n\n\n\n

Anti-diabetic\nstudies such as the \u03b1-amylase inhibitory assay and the \u03b1-glucosidase inhibitory\nassay were studied. <\/p>\n\n\n\n

\u03b1-amylase inhibitory assay<\/strong><\/p>\n\n\n\n

Based on the\nprotocol, ESENS and beta-caryophyllene of various concentrations were prepared\nand mixed with di-methyl sulfoxide solution (DMSO). Simultaneously, alpha-amylase\nwas prepared by mixing it with phosphate buffer (pH 6.8). The reaction mixture\nprepared was incubated, after which 1% starch solution was added to all the\ntubes. In addition, the tubes were again incubated for another 15 minutes. The\nreaction was stopped by adding 1ml of di-Nitro Salicylic acid (DNS) reagent,\nand the tubes were boiled in a water bath for 10 minutes. The contents were\nthen cooled, and 10 ml of distilled water was added to all the tubes.\nAbsorbance at 540 nm was measured with acarbose as a positive control. 20,21<\/sup>\n<\/p>\n\n\n\n

\u03b1-glucosidase inhibitory assay<\/strong><\/p>\n\n\n\n

\u03b1-glucosidase inhibitory assay was studied based on\nthe protocol 16<\/sup>. 112 \u03bcl of potassium phosphate buffer at pH 6.8 was\nmixed with 20 \u03bcl of enzyme solution, 8 \u03bcl of the extract, and\nbeta-caryophyllene and incubated for 15 minutes at 37\u00b0C. Following the\nincubation, 20 \u03bcl of NPG was added and again incubated for 15 minutes at 37\u00b0C.\nThe reaction was then terminated using 80 \u03bcl of Na2CO3 solution. The sample\nabsorbance was measured at 450 nm. For control, 8 \u03bcl of Dimethyl Sulfoxide\n(DMSO) was added to the reaction mixture instead of extract and compound.\nAcarbose serves as a standard.                                                                              \n<\/p>\n\n\n\n

Enzymatic and non-enzymatic assays of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

The enzymatic assays such as Acetyl cholinesterase\n(AchE) hydrolysis 22<\/sup> and the non-enzymatic assays such as Metal\nchelating assay 23<\/sup> were studied using the Ethanolic Seed Extract of Nigella\nsativa <\/em>(ESENS) and beta-caryophyllene to analyze the effectiveness of the\ncompound and the ESENS in preventing the hydrolysis of the enzyme AchE, which\nis a neurotransmitter.<\/p>\n\n\n\n

Enzymatic assay- Acetyl cholinesterase assay<\/strong><\/p>\n\n\n\n

The\nacetylcholinesterase assay of the Ethanolic Seed Extract of Nigella sativa <\/em>(ESENS)\nand the compounds were performed by adding 40 \u03bcl of 0.28 U\/ml acetylcholine\nenzyme and 140 \u03bcl of 3.3 mM 5,5-dithiobis-(2-nitrobenzoic) acid, which is\nprepared using a 0.1 M phosphate buffered solution of pH 7.0 that also contains\n6 mM of NaHCO3. The extract, the compounds of various concentrations, and 80 \u03bcl\nof phosphate buffered saline of pH 8.0 were added to the reaction mixture. The\nsolution was incubated for about 20 minutes at 25\u00b0C. 40 \u03bcl of 0.5 mM\nacetylthiocholine iodide was further added to each well containing the\nsolution. After the addition of substrate, the reaction mixture was measured at\n412 nm using UV-Vis spectrophotometry. <\/p>\n\n\n\n

Non-enzymatic assay \u2013 Metal chelating assay<\/strong><\/p>\n\n\n\n

To the mixture containing 168 \u03bcl of 0.1 M tris-HCL, 218 \u03bcl of 0.8% w\/v sodium chloride, 150 \u03bcl of 500 \u03bcM freshly prepared FeSO4, further, various concentrations of extract and compounds were added, followed by incubation for about 20 minutes. To the incubated mixture, 3 \u03bcl of 0.25% of 1, 10 – Phenanthroline was added to read the absorbance of the mixture at 510 nm.<\/p>\n\n\n\n

Results <\/strong><\/p>\n\n\n\n

Anti-oxidant activity of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n\n\n\n

2,2\nDiphenyl-1-Picrylhydrazyl (DPPH) radical scavenging assay:<\/strong><\/p>\n\n\n\n

Diphenyl-1-picrylhydrazyl (DPPH) is more stable for\nestimating free radicals. It is due to the ability to donate hydrogen\nmolecules. It takes up H molecules to form a dimagnetic compound, which is more\nstable. The ESENS showed good activity as compared to the control ascorbic\nacid, whose IC50 value is 11.26\u00b10.93\u03bcg\/ml and for ascorbic acid it is\n2.46\u00b10.41\u03bcg\/ml. Beta-caryophyllene exhibited good potential, and its IC50 value\nis 14.02\u00b10.71\u03bcg\/ml. This result suggests that ESENS and beta-caryophyllene\nexhibit good potential activity (Fig. 1,2). <\/p>\n\n\n\n\n\n
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Figure 1: DPPH radical scavenging activity of ESENS<\/strong><\/p>\n

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Figure 2: DPPH radical scavenging activity of compound beta-caryophyllene<\/strong><\/p>\n

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Hydrogen peroxide scavenging assay<\/strong><\/p>\n\n\n\n

The hydrogen peroxide activity exhibited is dose\ndependent when compared to the control Ascorbic acid, whose IC50 values are\nfound to be 17.50\u00b10.11\u03bcg\/ml for Ascorbic acid, 95.14\u00b10.66\u03bcg\/ml for\nbeta-caryophyllene. This reveals that the ESENS exhibited good radical\nscavenging activity when compared to the control where Beta caryophyllene\nexhibited less radical scavenging activity (Fig. 3,4).<\/p>\n\n\n\n\n\n
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Figure 3: Hydrogen peroxide radical scavenging assay of ESENS<\/strong><\/p>\n

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Figure 4: Hydrogen peroxide radical scavenging assay of compound beta-caryophyllene<\/strong><\/p>\n

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Nitric Oxide Radical scavenging assay <\/strong><\/p>\n\n\n\n

ESENS and beta-caryophyllene significantly inhibit\nnitric oxide in a dose-dependent manner at a concentration of 122\u00b10.11\u00b5g\/ml of\nESENS and 132\u00b10.21\u00b5g\/ml of beta-caryophyllene, respectively, when compared to\nascorbic acid. The result suggests that the extract and the compound\nbeta-caryophyllene are capable of inhibiting nitric oxide and proves that they\ncan be used as drugs in the indigenous system for treating various diseases and\ndisorders (Fig. 5,6).<\/p>\n\n\n\n\n\n
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Figure 5: Nitric oxide radical scavenging assay of ESENS<\/strong><\/p>\n

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Figure 6: Nitric oxide radical scavenging assay of compound beta-caryophyllene<\/strong><\/p>\n

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Anti-invitro\n<\/em>studies of ESENS and the compound beta-caryophyllene <\/strong><\/p>\n\n\n\n

HRBC membrane stabilization assay <\/strong><\/p>\n\n\n\n

Different concentrations of ESENS and\nbeta-caryophyllene were analyzed to study the prevention of hemolysis, which is\ndetermined by the HRBC membrane stabilization assay. Triton X 100 is a\ndetergent that destabilizes the RBC membrane and leaks hemoglobin, and the\nresults suggest that ESENS and beta-caryophyllene prevent the leaking of\nhemoglobin and prevent the integrity of the RBC; furthermore, it is proved to\nbe non-toxic in nature and can be used in biological studies (Fig. 7,8).\nIncreased levels of IL-1 beta cytokines are the major cause of various\nmetabolic disorders. The compound beta-caryophyllene is found to be more potent\nwhen compared to ESENS. <\/p>\n\n\n\n\n\n
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Figure 7: Membrane stabilization assay of ESENS.<\/strong><\/p>\n

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Figure 8: Membrane stabilization assay of compound beta-caryophyllene<\/strong><\/p>\n

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Cytotoxicity assay<\/strong> <\/p>\n\n\n\n

The isolated lymphocytes were treated with ESENS and\nbeta-caryophyllene to study the cell\u2019s toxicity. The ESENS exhibited 50% cell\ndeath, and beta-caryophyllene exhibited 40% cell death when compared to the\ncontrol sample (Table 1,2). This result suggests that the extract and compound\nexhibited less cell proliferation (Fig 9, 10).<\/p>\n\n\n\n

Table 1: Cytotoxicity assay of ESENS<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

INCUBATION TIME (HOURS)<\/strong><\/p>\n<\/td>\n

\n

IC50 VALUE (MG\/ML)- TRYPAN BLUE<\/strong><\/p>\n<\/td>\n

\n

IC50 VALUE (MG\/ML)-MTT<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

24<\/p>\n<\/td>\n

\n

6.83\u00b10.71a<\/sup><\/p>\n<\/td>\n

\n

8.07\u00b10.52*<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

48<\/p>\n<\/td>\n

\n

4.93\u00b10.25b<\/sup><\/p>\n<\/td>\n

\n

5.20\u00b10.10**<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

72<\/p>\n<\/td>\n

\n

3.70\u00b10.10c<\/sup><\/p>\n<\/td>\n

\n

4.47\u00b10.12**<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Table 2: Cytotoxicity assay of compound Beta caryophyllene<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

INCUBATION TIME (HOURS)<\/strong><\/p>\n<\/td>\n

\n

IC50 VLAUE (MG\/ML)- TRYPAN BLUE<\/strong><\/p>\n<\/td>\n

\n

IC50 VALUE (MG\/ML)-MTT<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

24<\/p>\n<\/td>\n

\n

6.93\u00b10.51a<\/sup><\/p>\n<\/td>\n

\n

10.07\u00b10.42*<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

48<\/p>\n<\/td>\n

\n

4.83\u00b10.45b<\/sup><\/p>\n<\/td>\n

\n

7.30\u00b10.20**<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

72<\/p>\n<\/td>\n

\n

2.90\u00b10.11c<\/sup><\/p>\n<\/td>\n

\n

6.37\u00b10.14**<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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Figure 9: Cell viability assay of ESENS<\/strong><\/p>\n

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Figure 10: Cell viability assay of compound beta-caryophyllene<\/strong><\/p>\n

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Enzyme linked immune-sorbent assay of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

The ESENS and the compound beta-caryophyllene in\ninhibition of IL-1 Beta were analyzed. The effects of ESENS and the compound\nbeta-caryophyllene are expressed in Fig. 11. Based on the result, it is\nsuggested that the compound has a high potency in inhibiting IL-1 Beta when\ncompared to the extract ESENS, which showed a good potential effect. The\nsamples are compared with the standard of IL-1 Beta and the lymphocyte cells\nthat are treated with hydrogen peroxide. The compounds have exhibited good\npotential effects individually when compared to the ESENS; it is revealed that\nthe compound beta-caryophyllene has anti-inflammatory activity.<\/p>\n\n\n\n\n\n
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Figure 11: Anti-inflammatory activity of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n

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Anti-diabetic activity of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n\n\n\n

\u03b1-amylase inhibitory assay<\/strong><\/p>\n\n\n\n

The\n\u03b1-amylase inhibitory assay of the ESENS and beta-caryophyllene was used to\nanalyze the inhibitory activity of the extract and the compound. The extract\nand the compound at various concentrations determined exhibited the highest\ninhibition rate of about 50.84% and the lowest inhibition rate of 15.49% for\nESENS and beta-caryophyllene, respectively, at 55.81% and 13.04%. The extract\nand the compound are compared with the control acarbose. Based on the results,\nthe compound beta-caryophyllene exhibited effective inhibitory activity when\ncompared to ESENS, and it showed dose-dependent activity (Fig. 12).<\/p>\n\n\n\n\n\n
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Figure 12: \u03b1-amylase activity of ESNS and the compound beta-caryophyllene<\/strong><\/p>\n

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\u03b1-glucosidase inhibitory assay<\/strong><\/p>\n\n\n\n

\u03b1-glucosidase\ninhibitory activity of ESENS and beta-caryophyllene was analyzed and determined\nusing the substrate p-NPG, which was then compared with the control acarbose.\nESENS and the compound beta-caryophyllene of various concentrations were\nstudied, and the result suggests that the compound beta-caryophyllene exhibited\n40% inhibition when compared to ESENS and the control. Based on the result, it\nis suggested that the compound beta-caryophyllene exhibited a good potential\neffect on the inhibitory action (Fig. 13). \n<\/p>\n\n\n\n\n\n
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Figure 13: \u03b1-glucosidase activity of ESENS and the compound beta-caryophyllene<\/strong><\/p>\n

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Enzymatic and non-enzymatic assay of ESENS and the compound\nbeta-caryophyllene<\/strong><\/p>\n\n\n\n

Enzymatic assay- Acetylcholinesterase assay<\/strong><\/p>\n\n\n\n

It is an in vitro<\/em> assay that is used to\nstudy the enzyme cholinesterase, which plays a major role in neurotransmittance\nin Alzheimer\u2019s disease. This reveals the role of ESENS and beta-caryophyllene,\nwhere beta-caryophyllene is 42.06\u00b12.1\u03bcg\/ml and ESENS is 84.7\u00b14.3\u03bcg\/ml. The\ncompound exhibited higher inhibition of the enzyme cholinesterase when compared\nto ESENS (Fig. 14,15).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 14: Enzymatic assay \u2013 Acetyl cholinesterase assay of ESENS VS drug Donepezil<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 15: Enzymatic assay \u2013 Acetyl cholinesterase assay of compound beta-caryophyllene VS drug Donepezil<\/strong><\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Non-enzymatic assay \u2013 Metal chelating assay<\/strong><\/p>\n\n\n\n

The results obtained from non-enzymatic assays\nsuggest that the ESENS of various concentrations chelates the ferric ion\n(Fe-II) and is found to exhibit 1.75\u00b10.17*<\/sup> at 50 \u00b5g\/ml when compared\nto the compound beta-caryophyllene of about 5.67\u00b10.53*<\/sup>. The results\nrevealed that the ESENS have high scavenging activity when compared to the\ncompounds. But Beta caryophyllene is found to have the same efficacy as\nbeta-caryophyllene (Table 3).<\/p>\n\n\n\n

Table 3: Non-Enzymatic assay \u2013 Metal chelating assay of ESENS and compound Beta Caryophyllene VS drug Donepezil<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

S.NO<\/strong><\/p>\n<\/td>\n

\n

PARAMETERS ASSESSED<\/strong><\/p>\n<\/td>\n

\n

NIGELLA SATIVA<\/em><\/strong><\/p>\n<\/td>\n

\n

BETA CARYOPHYLLENE<\/strong><\/p>\n

 <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

4.28\u00b10.31*<\/sup><\/p>\n<\/td>\n

\n

7.11\u00b10.93*<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

15<\/p>\n<\/td>\n

\n

3.12\u00b10.22*<\/sup><\/p>\n<\/td>\n

\n

6.04\u00b10.88*<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

25<\/p>\n<\/td>\n

\n

2.06\u00b10.21*<\/sup><\/p>\n<\/td>\n

\n

5.89\u00b10.67*<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

50<\/p>\n<\/td>\n

\n

1.75\u00b10.17*<\/sup><\/p>\n<\/td>\n

\n

5.67\u00b10.53*<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The tables should be marked with a number and a title. The figures must contain the captions. Figure\/Photos submitted must be of high resolution. Proper reference must be given for the figures and photos.<\/p>\n\n\n\n

The medicinal plants are\nused as the most abundant biosource for medication production. Numerous\npharmacological properties of Nigella sativa<\/em> have been discovered,\nincluding anti-analgesic, anti-ulcer, anti-inflammatory, anti-bacterial,\nanti-microbial, anti-cancer, and anti-diabetic properties. The qualitative\nanalysis of the ESENS revealed positive relevance in both therapeutic and\nphysiological activities, according to the current research. The isolation and\nidentification of these bioactive substances pave the way for the creation of\nnovel medications that may be used to treat a wide range of illnesses and\nailments. Tannins have medicinal benefits for anti-diarrhoea, anti-haemostatic,\nand a broad spectrum of anti-microbial activity against viruses, bacteria, and\nfungi. Alkaloids are the largest group of compounds that have been linked to\nmany medicinal properties for years and have a potential for cytotoxicity.\nSteroids have antibacterial properties, whereas flavonoids are hydroxylated\nphenolic compounds made from plants that have potent antioxidant and\nanti-cancer properties 24<\/sup>. Flavonoids also have a favorable response\nto microbial infection. Terpenoids are an example of an essential lipid that\nhas an aromatic flavor and performs activities including controlling growth and\ncolor. Metabolites of phenols exhibit biological features, including\nanti-apoptosis, anti-aging, and anti-inflammatory effects. It is a compound\nthat protects the heart and enhances endothelial health. The blood pressure is\nalso lowered and controlled by glycosides 25<\/sup>.<\/p>\n\n\n\n

Essential oils and other\nplant extracts antioxidant properties are gaining increased attention in\nscientific circles as well as in the food, cosmetics, and pharmaceutical\nsectors. By using recognized antioxidants like ascorbic acid in DPPH radical\nscavenging assays, hydrogen peroxide scavenging assays, and nitric oxide\nassays, researchers were able to assess the anti-radical or free radical\nscavenging capabilities of plant extracts or essential oils. Antioxidants’\nability to cause a decrease in absorbance at 517 nm was utilized to gauge their\npotential to lower DPPH radical generation 21<\/sup>. The ability to\ntransfer hydrogen is the justification. ESENS and the substance\nbeta-caryophyllene demonstrated effective nitric oxide scavenging abilities.\nThe substance showed promise for scavenging free radicals in the DPPH test as\nwell. According to research 26<\/sup>, beta-caryophyllene exhibited reduced\nactivity, and hydrogen peroxide was shown to be dosage-dependent.<\/p>\n\n\n\n

When compared to the\ncrude extract of Nigella sativa<\/em>, the in vitro<\/em> investigations showed\nthat the compound beta-caryophyllene has a large number of phytochemical elements\nthat are medicinally relevant. The isolated component beta-caryophyllene and\nthe crude extract of Nigella sativa<\/em> both exhibit anti-inflammatory and\nanti-diabetic properties, which were shown by the IL-1 beta test, alpha\namylase, and alpha glucosidase inhibitory assays. While the crude extract of\nNigella sativa<\/em> and the compound beta-caryophyllene (CRA) showed less\neffectiveness in hydrogen peroxide scavenging activity, the molecule\nbeta-caryophyllene is more efficient than ESENS in DPPH radical scavenging, the\nnitric oxide test, the acetyl cholinesterase assay, and the metal chelating\nassay. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The in vitro<\/em> studies revealed that the ESENS have many phytochemical properties that have been found to be medically significant. The compound beta-caryophyllene tends to have reduced hydrogen peroxide activity when compared to ESENS, and the compound beta-caryophyllene has potential anti-inflammatory, anti-diabetic, AchE, and metal chelating properties. Since the compound is more effective in vitro<\/em>, pharmacological studies have shown it to be successful and promising for the treatment of Alzheimer\u2019s disease, which needs an effective therapy to control brain functions and other complications. Due to these qualities, the compound beta-caryophyllene is a prospective candidate for the treatment of Alzheimer’s disease. The future prospective of the current study is to conduct biophysical, kinetic characterization and animal studies of the ESENS and the compound beta-caryophyllene.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

I feel thankful to acknowledge that Dr. M.G.R Educational and Research Institute (Deemed to be University) for providing me the workspace for my research. I thank Dr. K. Gomathi, Professor and Deputy Head, Department of Biotechnology, Dr. M.G.R Educational and Research Institute, Chennai for supporting my work and for guiding me.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors have no conflict of interest to declare. The co-author has seen and agreed with the contents of the manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

There is no funding Sources<\/p>\n\n\n\n

Reference <\/strong><\/p>\n\n\n\n

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