molecules Article Comprehensive Biological Potential, Phytochemical Profiling Using GC-MS and LC-ESI-MS, and In-Silico Assessment of Strobilanthes glutinosus Nees: An Important Medicinal Plant Marya Aziz 1 , Saeed Ahmad 1 , Umair Khurshid 1, *, Irfan Pervaiz 2 , Arslan Hussain Lodhi 3 , Nasrullah Jan 4,5 , Sameera Khurshid 6 , Muhammad Adeel Arshad 7 , Mohamed M. Ibrahim 8 , Gaber A. M. Mersal 8 , Fahaad S. Alenazi 9,10 , Ahmed Awadh Saleh Alamri 11 , Juwairiya Butt 12 , Hammad Saleem 13, * and Zeinhom M. El-Bahy 14 1 2 3 4 5 6 7 Citation: Aziz, M.; Ahmad, S.; Khurshid, U.; Pervaiz, I.; Lodhi, A.H.; Jan, N.; Khurshid, S.; Arshad, M.A.; Ibrahim, M.M.; Mersal, G.A.M.; et al. Comprehensive Biological Potential, 8 9 10 11 12 13 Phytochemical Profiling Using GC-MS and LC-ESI-MS, and In-Silico 14 Assessment of Strobilanthes glutinosus * Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan Department of Pharmacy, University of Chenab, Gujrat 50700, Pakistan Department of Pharmacology, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan Akson College of Pharmacy, Mirpur University of Science and Technology, Mirpur 10250, Pakistan Department of Pharmaceutics, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan Bahawalpur College of Pharmacy, Bahawalpur Medical and Dental College, Bahawalpur 63100, Pakistan Institute of Pharmacy, Faculty of Pharmaceutical and Allied Health Sciences, Lahore College for Women University, Lahore 54000, Pakistan Department of Chemistry, College of Science, Taif University, Taif 21944, Saudi Arabia Department of Pharmacology, College of Medicine, University of Hail, Hail 55473, Saudi Arabia Medical Education Unit, College of Medicine, University of Hail, Hail 55473, Saudi Arabia Medical Services, Ministry of Interior-Security Forces Hospital in Najran, Najran 66256, Saudi Arabia School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK Institute of Pharmaceutical Sciences (IPS), University of Veterinary and Animal Sciences (UVAS), Lahore 54000, Pakistan Department of Chemistry, Faculty of Science, Al-Azhar University, Nasr City 11884, Egypt Correspondence: umair.khurshid@iub.edu.pk (U.K.); hammad.saleem@uvas.edu.pk (H.S.) Nees: An Important Medicinal Plant. Molecules 2022, 27, 6885. https:// doi.org/10.3390/molecules27206885 Academic Editors: Irwin Rose Alencar Menezes, Henrique Douglas Melo Coutinho, Almir Gonçalves Wanderley and Jaime Ribeiro-Filho Received: 14 September 2022 Accepted: 10 October 2022 Published: 14 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Abstract: Plants of the genus Strobilanthes have notable use in folklore medicines as well as being used for pharmacological purposes. The present work explored the biological predispositions of Strobilanthes glutinosus and attempted to accomplish a comprehensive chemical profile through GC-MS of different fractions concerning polarity (chloroform and n-butanol) and LC-ESI-MS of methanolic extract by both positive and negative ionization modes. The biological characteristics such as antioxidant potential were assessed by applying six different methods. The potential for clinically relevant enzyme (α-amylase, α-glucosidase, and tyrosinase) inhibition was examined. The DPPH, ABTS, CUPRAC, and FRAP results revealed that the methanol fraction presented efficient results. The phosphomolybdenum assay revealed that the n-hexane fraction showed the most efficient results, while maximum metal chelation potential was observed for the chloroform fraction. The GC-MS profiling of n-butanol and chloroform fractions revealed the existence of several (110) important compounds presenting different classes (fatty acids, phenols, alkanes, monoterpenes, diterpenes, sesquiterpenoids, and sterols), while LC-ESI-MS tentatively identified the presence of 44 clinically important secondary metabolites. The n-hexane fraction exhibited the highest potential against α-amylase (497.98 mm ACAE/g extract) and α-glucosidase (605.85 mm ACAE/g extract). Significant inhibitory activity against tyrosinase enzyme was displayed by fraction. Six of the prevailing compounds from the GC-MS study (lupeol, beta-amyrin, stigmasterol, gamma sitosterol, 9,12-octadecadienoic acid, and n-hexadecanoic acid) were modelled against α-glucosidase and αamylase enzymes along with a comparison of binding affinity to standard acarbose, while three compounds identified through LC-ESI-MS were docked to the mushroom tyrosinase enzyme and presented with significant biding affinities. Thus, it is assumed that S. glutinosus demonstrated effective antioxidant and enzyme inhibition prospects with effective bioactive molecules, potentially opening the door to a new application in the field of medicine. Molecules 2022, 27, 6885. https://doi.org/10.3390/molecules27206885 https://www.mdpi.com/journal/molecules Molecules 2022, 27, 6885 2 of 19 Keywords: Strobilanthes glutinosus; antioxidant; enzyme inhibition; tyrosinase inhibition; GC-MS; LC-ESI-MS; docking 1. Introduction Medicinal plants have shown substantial medicinal and therapeutic benefits, due to which they are becoming important worldwide. Plants have become an object of ample importance in research as well as alternative medicinal therapy [1]. The rapidly increasing population and poverty in the developing world hamper this population from availing of high-priced pharmaceutical products. Medicinal plants are their main source for health care delivery. Around 70–80% of the developing world depends on conventional remedies obtained from medicinal plants [2]. Several novel compounds have been isolated from plants and have demonstrated unique and interesting biological activities [3]. The current research focus is to extract pharmacologically active compounds from natural provenance that can be helpful, particularly in the area of diseases that presently lack an effective medicinal therapy. There is a major shift of attention from modern medicine to parallel herbal systems, leading to a revival of alternative medicines [4]. According to an estimate, drugs derived from natural sources account for 20–25% of all drugs which are mentioned in the Pharmacopeia. Several medicinal plants are being employed for disease management without any modification [5]. The study of disease progression and induction has shown that oxidative stress is a major causative agent of various diseases. Chronic accumulation of reactive oxygen species causes cellular oxidative stress which ultimately leads to disease progression. Antioxidants of plant origin exhibit great potential; therefore, therapeutic focus has shifted towards the herbal medicine [6]. Phytochemicals possess great antioxidant activity that contributes to the therapeutic efficacy of plants [7]. Strobilanthes is a genus belonging to the family Acanthaceae, comprising around 350 species [8]. In this genus, the majority of plants present with anti-inflammatory and wound healing properties. These also show potential antimicrobial, anti-diabetic, and anticancer activities. Their extracts have been effective in spider poisoning, influenza epidemic, cerebrospinal meningitis, viral pneumonia, mumps, and acute respiratory syndrome [9]. Even though pharmacological research has described a broad variety of biological activities and chemical properties of the Strobilanthes genus (See Supplementary Materials), several species of the genus remain unexplored. S. glutinosus is a species that has not been studied scientifically in terms of biological and chemical properties. Only one study regarding the antimicrobial and antioxidant activity of this plant has been conducted in the Department of Botany, Mirpur University of Science and Technology (MUST), Mirpur-10250 (AJK), Pakistan [10]. Therefore, the current work was designed to conduct the chemical analysis to evaluate the bioactive content and GC-MS analysis of different extracts in order to analyze the phytochemical composition. The biological potential was studied by performing antioxidant assays of hydro-methanol extract and n-butanol, chloroform, and n-hexane fractions of the whole plant of S. glutinosus. Additionally, the present work proposed to determine the inhibitory effect of key enzymes (alpha-glucosidase and alpha-amylase) involved in diabetes mellitus along with molecular docking studies to explore any probable interaction between observed secondary metabolites and reported enzyme inhibition results. Molecular docking became an imperative tool for searching for inhibitor interactions at the receptor’s active site. Docking studies, to calculate binding free energy, also reveal the most appropriate confirmation that aids in the development of novel inhibitors against targeted enzymes. In terms of the literature evaluation, this study may be considered the preliminary analysis of the phytochemical composition, antioxidant properties, enzyme inhibition, and molecular docking studies of selected compounds from GC-MS of S. glutinosus. Molecules 2022, 27, 6885 3 of 19 2. Results 2.1. Phytochemical Composition In this recent work, two different extracts of S. glutinosus were assessed for their bioactive contents via GC-MS, as presented in Tables 1 and 2 and Figure 1, which enabled the tentative identification of 110 compounds. This GC-MS phytochemical investigation of different extracts of S. glutinosus can be considered the first comprehensive study. Table 1. GC-MS analysis of chloroform fraction of S. glutinosus. Sr. RT % Area Name of Compound Mol. Weight Mol. Formula Chem. Class 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3.07 3.14 3.37 10.41 14.12 14.33 15.02 15.07 17.27 17.90 18.18 27.56 27.97 28.18 28.47 28.64 29.37 29.97 30.82 31.02 31.12 32.35 33.00 33.24 33.84 35.92 36.65 37.28 37.89 0.69 4.14 1.75 0.27 0.29 0.90 0.27 3.57 0.94 2.40 0.41 0.34 0.44 0.47 1.33 0.74 1.18 5.53 2.42 0.85 0.74 0.43 0.77 0.76 2.29 5.74 0.46 2.52 4.58 Ethylbenzene Benzene, 1,3-dimethylp-Xylene Phenol, 2,5-bis(1,1-dimethylether 2-Pentadecanone, 6,10,14-trimet, . . . 9-Octadecene 2-Cyclopenten-1-one, 2-pentylHexadecanoic acid, methyl ester 9,12-Octadecadienoic acid, meth, . . . (R)-(-)-14-Methyl-8-hexadecyn-1-ol 2-Piperidinone, N-[4-bromo-n-bu, . . . Pyridine-3-carboxamide, oxime, . . . 2-Ethylacridine Cyclotrisiloxane, hexamethylEicosane Cholesta-6,22,24-triene, 4,4-di, . . . 1,3,5-Trisilacyclohexane, 1,1-d, . . . Cholest-5-en-3-ol (3.beta.)-, c, . . . Ergosta-4,6,22-trien-3.beta.-ol Phenylacetic acid, 2-(1-adamant, . . . Benz[b]-1,4-oxazepine-4(5H)-thi, . . . 2,4-Cyclohexadien-1-one, 3,5-bi, . . . 1H-Indole, 1-methyl-2-phenyl1-Bromoeicosane Campesterol Stigmasterol, 22,23-dihydrobeta.-Amyrin Lup-20(29)-en-3-one Lupeol 106 160 106.16 206.32 268.5 252.5 152.3 270.5 294.5 252.4 234.1 137.4 207.2 222.4 282.5 394.7 339.0 386.7 396.6 298.4 207.2 184.1 207.2 361.4 400.7 412.7 426.7 424.7 426.7 C8 H10 C8 H4 Cl6 C8 H10 C14 H22 O C18 H36 O C18 H36 C10 H16 O C17 H34 O2 C19 H34 O2 C17 H32 O C9 H16 BrNO C6 H7 N3 O C15 H13 N C6 H18 O3 Si3 C20 H42 C29 H46 C3 H6 Cl6 Si3 C27 H46 O C28 H44 O C2o H26 O2 C11 H13 NOS C12 H8 O2 C15 H13 N C20 H41 Br C28 H48 O C29 H48 O C30 H50 O C30 H48 O C30 H50 O Alkylbenzene Alkylbenzene Hydrocarbon Phenol Sesquiterpenoids Hydrocarbon Cyclic ketones Fatty acid Fatty acid Hydrocarbon Delta-lactams Oxime Acridine Organosilicon Alkane Sterol Hetrocyclic Cholesterol Sterol Ethyl ester Alkyl benzene Cyclohexadien Phenyl indole Alkane Sterol Sterol Triterpenoid Triterpenoid Triterpenoid Table 2. GC-MS analysis of n-butanol fraction of S. glutinosus. Sr. RT % Area Name of Compound Mol. Weight Mol. Formula 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 3.06 3.13 3.37 6.63 7.40 7.60 8.94 9.77 10.19 10.42 10.71 11.30 11.38 11.93 12.08 0.13 1.31 0.64 0.03 0.01 0.01 0.02 0.02 0.05 0.09 0.01 0.03 0.06 0.05 0.02 Ethylbenzene p-Xylene o-Xylene m-Mentha-4,8-diene, (1S,3S)-(+)1H-Inden-1-one, 2,3-dihydro-3,4, . . . Decane, 3,8-dimethyl1-Tetradecene Nonadecane Pentacosane Phenol, 2,5-bis(1,1-dimethyleth, . . . Octacosane 1-Hexadecene Hexadecane 2-Undecene, 5-methylHexadecane, 2-methyl- 106.1 106.1 106.1 136.2 174.2 170.3 196.3 268.5 352.7 206.3 394.8 224.4 226.4 168.32 240.5 C8 H10 C8 H10 C8 H10 C10 H16 C12 H14 O C12 H26 C14 H28 C19 H40 C20 H52 C14 H22 O C28 H58 C16 H32 C16 H34 C12 H24 C17 H36 Chem. Class Ar. Ar. Ar. Ar. hydrocarbon hydrocarbon hydrocarbon hydrocarbon Indanones Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Phenol Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Molecules 2022, 27, 6885 4 of 19 Table 2. Cont. Sr. RT % Area Name of Compound Mol. Weight Mol. Formula Chem. Class 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 12.18 12.49 12.56 12.63 12.97 13.18 13.27 13.51 13.70 14.12 14.16 14.24 14.54 14.66 14.84 15.07 15.12 15.36 15.58 15.90 16.48 16.73 16.91 16.98 17.46 17.55 17.69 18.39 18.50 19.19 19.31 19.97 20.22 20.28 20.35 20.43 20.61 20.69 21.41 21.64 21.80 21.91 22.03 22.66 22.95 23.07 23.45 23.54 23.71 24.05 24.12 24.19 24.26 24.73 24.85 24.91 26.12 26.24 0.03 0.16 0.08 0.04 0.05 0.04 0.03 0.03 0.14 0.02 0.03 0.02 0.05 0.05 0.02 0.13 0.02 0.02 0.02 0.03 0.30 0.04 0.08 0.11 0.14 0.12 0.16 0.17 0.21 0.72 0.19 0.24 0.33 0.23 0.32 0.40 0.81 0.24 0.21 1.01 0.38 0.74 1.06 1.01 0.99 1.24 1.46 0.45 1.05 0.32 0.46 0.26 1.01 0.77 1.41 0.79 0.74 2.61 Pentadecane Heptadecane Pentadecane, 2,6,10,14-tetramet, . . . Hentriacontane Tetratetracontane Heptadecane, 2-methylHeptadecane, 3-methyl1-Octadecene Hexadecane, 2,6,10,14- phytane) 7-Oxabicyclo [4.1.0]heptane, 1,5, . . . Tetradecane, 5-methylPentadecane Tetrapentacontane, 1,54-dibromoNonadecane, 9-methylCyclotetradecane, 1,7,11-trimet, . . . Pentadecanoic acid, 14-methyl-, . . . 7,9-Di-tert-butyl-1-oxaspiro(4, . . . Octadecane, 1-chloroCyclopentadecane 1-Nonadecene Heneicosane Octadecane Nonadecane Cycloeicosane 1-Docosene 2-Eicosanol, (.+ /− .)tert-Hexadecanethiol Tridecane, 6-cyclohexylHexadecanoic acid, butyl ester Nonahexacontanoic acid Nonadecane, 1-chloroDocosane Tricosane Cyclotetradecane, 1,7,11-trimet, . . . Nonadecane, 1-chloro1-Chloroeicosane Docosane Octadecane Hexadecane, 1-iodo1-Chloroeicosane 1-Tricosene 1-Nonadecene Hexadecane, 1-iodo1-Hexacosene Pentacosane Hexacosane Nonadecane, 9-methylHexadecane, 2-methylDi-n-octyl phthalate Nonahexacontanoic acid Ethanol, 2-(octadecyloxy)1-Chloroeicosane Octadecane 1-Decanol, 2-hexylNonadecane, 9-methylOctadecane, 1-iodoTricosane Heptacosane, 1-chloro- 212.4 240.5 268.5 436.8 619.2 215.4 254.9 252.6 282.5 194.2 212.4 212.4 917.2 282.5 280.5 256.4 276.4 288.9 210.4 266.5 296.6 254.5 268.5 280.5 308.6 298.5 258.2 266.5 312.5 999.8 303 310.6 324.6 280.5 302.9 317.0 310.6 254.5 352.34 317.0 322.6 266.5 352.34 364.7 352.7 366.71 282.5 240.5 390.6 999.8 314.5 317.0 254.4 242.44 282.5 380.4 324.6 415.2 C15 H32 C17 H36 C19 H40 C31 H64 C44 H90 C18 H38 C18 H38 C18 H36 C20 H42 C12 H18 O2 C15 H32 C15 H32 C54 H108 Br2 C20 H42 C20 H40 C16 H32 O2 C17 H24 O3 C18 H37 Cl C15 H30 C19 H38 C21 H44 C18 H38 C19 H40 C20 H40 C22 H44 C20 H42 O C16 H34 S C19 H38 C20 H40 O2 C69 H138 O2 C19 H39 Cl C22 H46 C23 H48 C20 H40 C19 H39 Cl C20 H41 Cl C22 H46 C18 H38 C16 H33 I C20 H41 Cl C23 H46 C19 H38 C16 H33 I C 26 H52 C25 H52 C26 H54 C20 H42 C17 H36 C24 H38 O4 C69 H138 O2 C20 H42 O2 C20 H41 Cl C18 H38 C16 H34 O C20 H42 C18 H37 I C23 H48 C27 H55 Cl Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Diterpene Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Diterpene Fatty acid Flavanoids Alkyl chloride Alkane Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Ali. hydrocarbon Alkane Ali. hydrocarbon Phenol Thiol Ar. hydrocarbon Fatty acid ester Fatty acid Alkane Alkane Alkane Alkane Alkane Alkyl halide Alkane Alkane Alkyl halide Alkyl halide Alkene Alkene Alkyl Halide Alkene Alkane Alkane Alkane Alkane Benzoic acid esters Fatty acid Phenol Alkyl halide Alkane Alchohol Alkane Alkyl halide Alkane Alkyl halide Molecules 2022, 27, 6885 5 of 19 Table 2. Cont. Sr. RT % Area Name of Compound Mol. Weight Mol. Formula Chem. Class 74 75 76 77 78 79 80 81 27.21 27.58 28.89 31.40 33.24 34.60 35.49 36.25 1.36 2.56 3.64 2.01 0.45 0.09 0.08 0.11 Heptacosane Octacosane Eicosane Heneicosane, 3-methyl1-Bromoeicosane Z-14-Nonacosane Methoxyacetic acid, heptadecyl, . . . Tetratriacontane, 17-hexadecyl- 380.7 394.7 282.5 310.6 361.4 406.8 314.5 703.3 C27 H56 C28 H58 C20 H42 C22 H46 C20 H41 Br C29 H58 C19 H38 O3 C50 H102 Alkane Alkane Ali. hydrocarbon Alkane Alkyl Halide Alkanes Ester Alkanes Figure 1. GC-MS chromatograms of chloroform (A) and n-butanol (B) fractions. To gain a more in-depth insight into the phytochemical composition through the LC-ESI-MS method, we looked into the phytochemicals present in the plant S. glutinosus in greater detail. Due to its many benefits, including low solvent consumption, high precision, and accuracy, the hybrid coupled technique is frequently employed for the investigation of phytochemicals derived from plants [11]. Positive and negative ionizing modes of LC-ESI-MS-MS were used to monitor the profile of secondary metabolites, resulting in the identification of 44 compounds (Tables 3 and 4). Phenols, phenolic acids, phenolic glycosides, flavonoids, flavonoid glucoside, fatty acids, triterpenoids, lignans, and coumarin are just some of the chemical classes represented Molecules 2022, 27, 6885 6 of 19 by the compounds identified. Figures 2 and 3 represents total ion chromatograms of both negative and positive ionization modes. ratensein 7 β glucoside 6″ Table 3. LC-ESI-MS-MS screening of Strobilanthes glutinosus in negative mode. Sr. RT (min) % Area Tentative Identification Mol. Formula Mol. Mass Adduct Chemical Class 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 10.95 11.70 11.91 12.25 12.46 12.70 2.21 13.06 2.37 13.32 2.63 13.40 13.61 14.09 14.29 14.49 14.71 15.01 15.99 0.49 0.22 1.57 0.36 5.84 0.47 0.55 2.74 0.33 1.15 0.25 0.68 0.94 1.28 1.16 0.93 1.58 0.33 1.88 C9 H6 O4 C10 H9 NO C9 H10 O5 C15 H10 O4 C16 H12 O6 C16 H22 O4 C13 H12 O7 C18 H34 O2 C15 H14 O6 C17 C26 O4 C20 H20 O5 C13 H12 O9 C15 H18 O8 C20 H20 O7 C19 H20 O8 C28 H48 O C30 H50 O C22 H18 O10 C20 H18 O12 177 159 199 253 255 277 279 281 289.5 293.50 295.00 311.00 325.00 371.00 377 400 425 441.50 449.50 [−H] [−H] [−H] [−H] [−H] [−H] [−H] [−H] [−H] [−H] [HCOO] [−H] [−H] [−H] [−H] [−H] [−H] [−H] [−H] Coumarin Other Phenol Flavonoid Flavonoid Terpenoid Phenol Fatty acid Phenol Phenol Flavonoid Phenolic acid Phenolic acid Lignan Phenol Terpenoid Terpenoid Flavonoid Flavonoid 20 16.16 1.46 C21 H24 O11 451.15 [−H] Glucoside 21 16.34 4.32 C30 H36 O10 555.00 [−H] Lignan 22 16.76 1.76 C25 H23 O13 549.50 [−H] Flavonoid 23 17.05 0.49 Aesculetin Echinospine Syringic acid Daidzein Hispidulin Emmotin A p-coumaryl malic acid Oleic acid Catechin Gingerol 8-Prenylnaringenin Caffeoyl tartaric acid p-coumaric acid hexoside Sesamolinol Oleuropein aglycone α Campesterol Beta-amyrin (−)-Epicatechin 3-O-gallate Myricetin 3-O-arabinoside 3-Hydroxyphloretin 2′ -O-glucoside Lariciresinol-sesquilignan 6,4′Pratensein 7-O-β-Dglucoside 6”O-malonate Luteolin 7- rutinoside C27 H30 O15 593 [−H] Flavonoid Figure 2. LC-ESI-MS-MS full scan of Strobilanthes glutinosus (negative mode) 50–400 (A), 50–800 (B), – – – and 50–2000 (C). Molecules 2022, 27, 6885 7 of 19 Table 4. LC-ESI-MS-MS screening of Strobilanthes glutinosus in positive mode. Sr. RT (min) % Area Tentative Identification Mol. Formula Mol. Mass Adduct Chemical Class 1 2 3 4 5 6 7 8 9 1.50 2.28 2.71 3.06 0.57 0.95 1.24 3.43 3.60 1.36 0.94 1.44 2.57 0.66 0.35 0.62 0.68 0.44 Betaine Gentesic acid Azelaic acid Angustifoline Apigenin Linoleinic acid Linoleic acid Eriodictyol Catechin C5 H11 NO2 C7 H6 O4 C9 H16 O4 C14 H22 N2 O C15 H10 O5 C18 H30 O2 C18 H32 O2 C15 H12 O6 C15 H14 O6 118.00 156.00 189.00 235.00 271.00 279.17 281.50 289.00 291.00 [+H] [+H] [+H] [+H] [+H] [+H] [+H] [+H] [+H] 10 3.98 0.27 Gallic acid hexoside C13 H16 O10 331.50 [+H] 11 12 13 4.28 5.90 6.21 0.38 1.68 1.74 7dehydro cholesterin α-tocopherol 5-OH liquiritin C27 H44 O C29 H50 O2 C21 H22 O10 385 429.30 435.30 [+H] [+H] [+H] 14 6.99 2.89 Ligstroside C25 H32 O12 523.30 [+H] 15 16 17 18 6.42 7.33 7.57 7.87 2.33 1.46 5.18 4.78 C21 H18 O12 C22 H26 O11 C30 H48 O10 C27 H30 O16 463.30 467.50 567.50 611.20 [+H] [+H] [+H] [+H] 19 8.32 4.56 C21 H20 O11 655.50 [+H] Flavonoid 20 8.84 7.23 C31 H28 O15 743.55 [+H] Flavonoid 21 9.97 5.99 Scutellarin Agnuside di-O-acetyldarutoside Rutin Quercetin-6,4′ -dimethoxy-3fructo-rhamnoside Quercetin rhamnoside-feruloyl-hexoside Quercetin 3-O-rhamnosyl-glucoside 7-O-rhamnoside Amino acid Phenolic acid Dicarboxylic acid Alkaloid Flavonoid Fatty acid Fatty acid Flavonoid Phenol Phenolic glycoside Terpenoid Terpenoid Flavonoid Phenolic glycoside Flavonoid Other Phenol Flavonoid C27 H30 O16 875.50 [+H] Flavonoid Figure 3. LC-ESI-MS-MS full scan of Strobilanthes glutinosus (positive mode) 50–400 (A), 50–800 (B), – – – and 50–2000 (C). Molecules 2022, 27, 6885 8 of 19 2.2. Antioxidant Assays In the present study, six different methods (DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, and metal chelating assays) were used to determine the antioxidant potential of S. glutinosus, and Table 5 furnishes the results of the study. Table 5. Antioxidant results by different methods of S. glutinosus whole plant extract/fractions. Radical Scavenging Assays Reducing Power Assays Extract/Fractions Methanol n-butanol Chloroform n-hexane Total Antioxidant Capacity Ferrous Ion Chelation DPPH (mg TE/g Extract) ABTS (mg TE/g Extract) CUPRAC (mg TE/g Extract) FRAP (mg TE/g Extract) Phosphomolybdenum (mg TE/g Extract) Metal Chelation (mg EDTAE/g Extract) 56.217 ± 0.66 a 47.920 ± 0.166 c 50.130 ± 0.108 b 9.804 ± 1.234 d 63.469 ± 0.045 a 47.669 ± 0.078 c 53.574 ± 5.183 b 12.761 ± 0.045 d 245.116 ± 4.240 a 162.629 ± 6.372 b 84.693 ± 2.780 c 59.878 ± 4.865 d 87.126 ± 0.083 a 75.097 ± 0.054 b 65.007 ± 0.361 c 34.971 ± 1.820 d 96.015 ± 0.476 b 6.544 ± 0.748 d 60.698 ± 0.079 c 119.587 ± 0.555 a 17.038 ± 0.0769 b 7.692 ± 0.0769 d 12.384 ± 8.423 c 25.346 ± 0.192 a All of the procedures were carried out thrice. The mean ± standard deviation were used to represent the results. Trolox and EDTAE were utilized as standard. Significantly different results were exhibited when compared to standard (p < 0.05). Superscripts (a–d) represents statistical difference. 2.3. In Vitro Enzyme Inhibition Activity The studied plant extracts were tested against different enzymes, including α-amylase, α-glucosidase, and tyrosinase. The standard used for α-amylase and α-glucosidase was acarbose, and the results were presented in mmol ACAE/g extract. Kojic acid was used as the standard for tyrosinase enzyme, with results being presented in mg KAE/g extract. The inhibitory potential of plant extract/fractions against all three enzymes is displayed in Table 6. Maximum percentage inhibition against α-amylase and α-glucosidase was displayed by chloroform fraction (501.407 ± 2.982 and 605.854 ± 6.252 mmol ACAE/g extract), respectively. While methanolic extract exhibited the highest potential against tyrosinase enzyme (9.86 ± 1.41 mg KAE/g extract). Table 6. Enzyme inhibition results of S. glutinosus whole plant extract/fractions. Extract/Fractions α-Amylase (mmol ACAE/g Extract) α-Glucosidase (mmol ACAE/g Extract) Tyrosinase (mg KAE/g Extract) Methanol n-butanol Chloroform n-hexane 166.758 ± 1.72 b 107.007 ± 1.104 c 501.407 ± 2.982 a 85.859 ± 0.510 d 294.195 ± 3.036 b 118.64 ± 1.224 c 605.854 ± 6.252 a 93.572 ± 0.965 d 9.86 ± 1.4 a 8.52 ± 1.82 b 6.91 ± 1.35 c NA All of the procedures were carried out thrice. The mean ± standard deviation were used to represent the results. ACAE: acarbose equivalent, KAE: kojic acid equivalent. Significantly different results were exhibited when compared to standard (p < 0.05). NA (No Activity). Superscripts (a–d) represents statistical difference. 2.4. In Silico Analysis A total of 29 compounds identified in GC-MS analysis of the chloroform fraction were docked, and six of them were selected based on their binding affinities along with the standard compound acarbose against the receptor α-glucosidase and α-amylase enzymes. PubChem, the drug database, was used for downloading the 3D structure of ligand molecules. Beta-amyrin, sitosterol, stigmasterol, and lupeol were identified to be the most suitable ligands, with significant binding affinities. Our results indicated that beta amyrin had the highest binding affinity with the α glucosidase enzyme, with a docking score of –8.4 kcal/mol, followed by stigmasterol (−7.5 kcal/mol), sitosterol (−7.5 kcal/mol), lupeol (−6.9 kcal/mol), 9,12-octadecadienoic acid (−4.1 kcal/mol), and n-hexadecanoic acid (−3.5 kcal/mol), presented in Table 7 and Figures 4 and 5. Beta amyrin, sitosterol, and stigmasterol presented the highest binding affinity versus α glucosidase enzyme, with a binding energy of −8.4 and −7.5 kcal/mol, respectively, ranking higher in comparison to standard drug acarbose (−6.6 kcal/mol). Molecules 2022, 27, 6885 9 of 19 Table 7. Binding affinities and interactions of ligands against (anti-diabetic) enzymes. Enzyme Ligands Binding Energy (kcal/mol) Electrostatic/Hydrophobic Interaction −6.6 Hydrogen bond (Thr448 , Asn443 , Ala514 , Asp441 , Glu432 , Arg437 , His348 ) C-H bond (Gln438 ) Lupeol −6.9 Alkyl interaction (Lys398 , Trp394 , Val380 , Trp354 ) Hydrogen bond (Ala229 , Asn301 ) Van-dar walls (Pro230 , Arg340 , Phe357 , Ala378 , Gly402 , Glu377 , Val335 , Leu227 , Met302 , Glu396 , Asp379 , Glu231 ) Sitosterol −7.5 Alkyl interaction (Leu45, Ala444) C-H bond (Leu433) Van-dar walls (Ala434, Arg450, Met407, Thr445) 9,12-octadecadienoic acid −4.1 Alkyl interaction (Leu446 ) Van-dar walls (Asp411 , Thr410 , Leu373 , Leu45 , Asp440 , Ser44 , Gln438 , Pro408 , Glu432 , Leu431 ) β-Amyrin −8.4 Alkyl interaction (Pro230 ) Hexadacanoic acid −3.9 Hydrogen bond (Aal380 ) C-H bond (Lyc398 , Gly998 ) Alkyl interaction (Val335 ) Stigmasterol −7.5 Alkyl interaction (Val335 , Ala343 , Met302 , Val334 , Phe397 , Phe297 ) Lupeol −7.6 Pi-sigma (Tyr59 ) Pi-alkyl (Trp60 ) Sitosterol −5.1 Pi-alkyl (Pro230 , Phe397 ) 9,12-octadecadienoic acid −4.9 Hydrogen bond (Asn273 ) Alkyl interaction (Tyr59 , Leu142 , Met302 , Ala177 , Leu141 ) β-Amyrin −8.4 Hydrogen bond (Asp274 ) Pi-sigma (Phe105 ) Pi-alkyl (Tyr59 ) Hexadacanoic acid −4.6 Van-dar walls (Asp274 , Tyr62 , Ala177 , Asp176 , Gln63 , Asp269 , Leu144 , Asn273 ) Pi-alkyl (Tyr59 , Trp58 , Leu142 ) C-H bond (His102 , Gln208 ) Stigmasterol −9.1 Pi-sigma (Tyr59 ) Pi-alkyl (Tyr59 , Leu142 , Phe105 ) Acarbose α-glucosidase α-amylase Molecular docking on three key compounds (Lingstroside, Rutin, and Scutellarin) identified from the methanolic extract using LC-ESI-MS (Figure 6) was performed against mushroom tyrosinase enzyme. As a reference drug for such conditions, kojic acid was also included in the assay. The binding affinities and amino acid interactions are presented in Table 8 and Figure 7. − Molecules 2022, 27, 6885 − 10 of 19 Enzyme α β Figure 4. Enzyme α-glucosidase and ligands interaction. Hexadecanoic acid (A), Lupeol (B), βAmyrin (C), Octadecadienoic acid (D), Sitosterol (E), Stigmasterol (F), Acarbose (standard) (G). Enzyme α-amylase α β Figure 5. Enzyme and ligands interaction. Hexadecanoic acid (A), Lupeol (B), β-Amyrin (C), Octadecadienoic acid (D), Sitosterol (E), Stigmasterol (F). Molecules 2022, 27, 6885 Enzyme α β 11 of 19 Figure 6. Molecular docking of selected ligands with tyrosinase enzyme. (A) Ligstroside, (B) Rutin, (C) Scutellarin, and (D) Kojic acid. Table 8. Binding affinities and interactions of the selected ligands from S. glutinosus extract by LC-ESI-MS against tyrosinase enzyme. Enzyme Ligand Binding Affinity (Kcal/mol) Amino Acids Interactions −8.0 Unfavorable Accaptor: (TYRA352 ) Pi Sigma: (VALA366 ) Conventional Hydrogen Bond: (ASNA15 , GLNA294 , GLYA360 , GLYA361 ) Carbon Hydrogen: (SERA351 , PHEA355 ) −8.9 Amide-Pi Stacked: (PHEA355 ) Pi-Alkyl: (PROA298 , LYSA359 ) Conventional Hydrogen Bond: (GLNA294 , THRA345 , VALA358 , GLYA360 , GLYA361 ) A13 Van der Waals: (VAL , GLYA299 , VALA300 , THRA343 , ASPA344 , ALAA346 , SERA351 , TYRA352 , PROA363 , VALA366 ) Scutellarin −8.6 Pi-Pi Stacked: (PHEA355 ) Pi-Alkyl: (ALAA295 , PROA298 ) Conventional Hydrogen Bond: (GLNA294 ) Carbon Hydrogen: (THRA343 ) Van der Waals: (SERA291 , TYRA297 , GLYA299 , VALA300 , TRPA301 , THRA345 , SERA351 , TYRA352 , PROA363 , VALA366 ) Kojic acid (Standard) −5.3 Pi-Pi Stacked: (PHEA355 ) Ligstroside Rutin Tyrosinase − Molecules 2022, 27, 6885 − 12 of 19 Figure 7. Enzyme tyrosinase and ligands interaction. Lingstroside (A), Rutin (B), Scutellarin (C). 3. Discussion Bioactive chemicals such as those found in plants are crucial to human health because they stimulate cell division and repair, two processes essential to being healthy as a whole [12]. There is evidence in the literature that indicates S. glutinosus methanol extract has significant flavonoid and phenolic levels [10]. Overall, 29 and 81 major compounds were identified from chloroform and n-butanol fractions, respectively. From 110 compounds, n-hexadecanoic acid, 9,12-Octadecadienoic acid (2,2)-, methyl ester, linoelaidic acid, 11,13-dimethy1-12-tetradecane-1-olacetate, heptadecanal, alpha-tocospiro A, alphatocospiro B, dl- stigmasterol, gamma–sitosterol, lup-20(29)-en-3-one, lupeol, and linoleic acid were identified as major bioactive compounds from the whole-plant extract. Lupeol (sterol) is one of the major compounds detected in high concentrations in the GC-MS study. Lupeol is found naturally in edible fruits and vegetables and is reported to have antioxidant, anti-diabetic, hepatoprotective, anti-inflammatory, anti-protozoal, anti-microbial, anti-proliferative, and cholesterol-lowering effects [13]. Another compound having a reported anticancer activity and antioxidant effect is 9, 12-Octadecenoic acid methyl ester (Z, Z), fatty acid methyl ester [14]. The pharmacological benefits of flavonoids and phenols are well-documented. Many studies have shown that phenols and flavonoids are effective antioxidants, anti-inflammatory agents, and enzyme inhibitors, with significant clinical uses [15–18]. In the present study (LC-ESI-MS), a total of 14 flavonoids were identified, 6 in negative mode and 8 in positive mode of ionization. Deprotonated molecules [M-H]− were observed in Daidzein (Rt = 12.25 min), Hispidulin (Rt = 12.46), 8-Prenylnaringenin (Rt = 2.63), (−), Epicatechin 3-O-gallate (Rt = 15.01), Myricetin 3-O-arabinoside (Rt = 15.99), and Luteolin 7- rutinoside (Rt = 17.05), while Apigenin (Rt = 0.57 min), Eriodictyol (Rt = 3.43), 5-OH liquiritin (Rt = 6.21), scutellarin (Rt = 6.42), and Rutin (Rt = 7.87) displayed ions’ positive mode of ionisation. Similarly, 10 phenols and their derivatives were tentatively identified in both the negative and positive mode of ionisation analysis. Syringic acid (Rt = 11.91), p-coumaryl malic acid (Rt = 2.21), catechin (Rt = 2.37), and gingerol (Rt = 13.32) are the phenols found, and all of them showed deprotonated molecules [M-H]− . In the present chromatographic analysis of the S. glutinosus methanol fraction, three phenolic acids, caffeoyl tartaric acid (Rt = 13.40), p-coumaric acid hexoside (Rt = 13.61), and gentesic acid (Rt = 2.28), were Molecules 2022, 27, 6885 13 of 19 tentatively identified. Additionally, catechin was detected in the positive ionisation mode, where it showed a peak at m/z 291.00. A single phenolic glycoside, gallic acid hexoside (Rt = 3.98), was tentatively identified in the positive mode of ionization by displaying the protonated molecule [M+H]+ at 331.50. In the process, two lignans, sesamolinol (Rt = 14.09) and lariciresinol-sesquilignan (Rt = 16.34), were isolated in the negative mode of the analysis. Emmotin A, a terpenoid (Rt = 12.70), was detected in the negative mode, as were campesterol (Rt = 14.49) and beta amyrin (Rt = 14.71), while tocopherol (Rt = 5.90) was detected in the positive mode. The oxidative stress caused by reactive oxygen species has been linked to the pathogenesis of a wide variety of degenerative illnesses [19]. Endogenous antioxidants with exogenous antioxidants mostly derived from plants prevent the oxidative stress [20] Plants are the main source of antioxidant compounds. Consequently, for oxidant-induced diseases, research has been focused on plants [19]. Antioxidant effects cannot be established by using a single method since plant extracts contain a large number of chemicals that comprise antioxidant activity with a varied mechanism of action [21]. For these reasons, different methods were applied to analyze the antioxidant activity of plant extracts. The hydro-methanol extract of S. glutinosus expressed the highest value in the DPPH assay (56.21 mg TE/g extract) and ABTS assay (63.46 mg TE/g extract) TE/g extract. The n-butanol and chloroform fractions shared almost similar results in both assays. whereas the lowest effect was measured towards the n-hexane fraction (DPPH: 9.80 mg TE/g extract) and (ABTS: 12.76 mg TE/g extract). The extract’s antioxidant activity is also significant because of its reducing power. By reducing the ferric tripridyltriazine complex to the ferrous complex at low pH, the FRAP assay measures the antioxidant’s capacity to contribute electrons to reduce ferric ions. The hydro-methanol extract presented higher reducing power (87.12 mg TE/g extract), while the n-hexane fraction exhibited the lowest reducing potential (34.97 mg TE/g extract). The n-butanol and chloroform fractions shared almost similar results, i.e., 75.09 and 65.00 mg TE/g extract, respectively. In the results for the CUPRAC assay, the hydro-methanol extract exhibited higher values (245.11 mg TE/g extract) following the order from hydro-methanol > n-butanol > chloroform > n-hexane. There was a link between free radical scavenging assays and reducing power assays, suggesting that the bioactive content results verified the increased amount of phenolic and flavonoid components in methanol and butanol extracts [22]. Furthermore, a phosphomolybdenum assay was used to evaluate the total antioxidant capacity, and the findings are furnished in Table 2. The n-hexane fraction of S. glutinosus exhibited the highest total antioxidant capacity (119.58 mg TE/g extract), while hydromethanol extract and chloroform fraction exhibited considerable total antioxidant capacity potential, with values of 96.01 and 60.69 mg TE/g extract, respectively. The n-butanol fraction was the least active fraction for this assay (6.54 mg Trolox equivalent/g). The presence of non-phenolic compounds with chelating properties among phytoconstituents is consistent with other findings reported by [23]. The GC-MS study of S. glutinosus presented the compounds thymol, lupeol, alpha-tocopherol, and squalene, having antioxidant activity reported by [24–26], which justifies the results. In developing nations, where Type 2 diabetes mellitus accounts for 90% of all cases, the prevalence of diabetes mellitus is anticipated to more than double, from 171 million in 2000 to 300 million by 2025 [27]. Popular antidiabetic drugs such as acarbose, voglibose, and miglitol all work by inhibiting alpha-amylase and alpha-glucosidase enzymes, resulting in lower blood glucose levels. However, these drugs have undesirable side effects, including toxicity to the liver and gastrointestinal issues, when used long-term [28]. Consequently, there is a demand for novel alpha-glucosidase and alpha-amylase inhibitors derived from natural origins, particularly from herbs and plants that produce no unpleasant or undesirable side effects in diabetic patients. The results of alpha-amylase, alpha-glucosidase, and tyrosinase inhibition assays of different fractions of S. glutinosus are presented in Table 6. Among the tested extracts, Molecules 2022, 27, 6885 14 of 19 chloroform fraction was most efficient against α-amylase (501.407 ± 2.98 mmol ACAE/g extract) and α-glucosidase enzyme (605.854 ± 6.252 mmol ACAE/g extract). Likewise, the hydro-methanol extract was also noticeably active against both (amylase and glucosidase) enzymes, with the values of 166.758 ± 1.721 and 294.195 ± 3.036 mmol ACAE/g extract, respectively. The evaluated anti-diabetic potential of S. glutinosus complies with the earlier reports as described in [29], an in vivo assay of S. cuspidata to evaluate the anti-diabetic potential and the isolated compounds. Lupein, (3-Hydroxy-4-methoxy phenyl) cinnamic acid and stigmasterol exhibited confirmed antidiabetic potential by inhibiting α amylase enzyme [29]. Melanin biosynthesis, also called melanogenesis, is a physiological process that is catalysed in humans by the enzyme tyrosinase [30]. Tyrosinase inhibitors may be useful for treating dermatological disorders associated with melanin hyperpigmentation [31], as they work by reducing the activity of tyrosinase, an enzyme that is responsible for the production of melanin. Tyrosinase inhibition can also be useful for the food industry. However, preventing tyrosinase activity is ideal for preserving the freshness of fruits and vegetables for a longer period of time. The methanolic extract showed prominent activity against the tyrosinase enzyme with a value of 9.86 ± 1.41 mg KAE/g extract. The tyrosinase inhibition results of S. glutinosus extracts were ordered as follows: methanol > n-butanol > chloroform > n-hexane. Studies have shown that various phenolics and flavonoids (as revealed via LC-ESI-MS of S. glutinosus methanol extract) have anti-tyrosinase potenial. To accurately anticipate the ligand–target binding energy and to offer an understanding of the molecular-based mechanism of biological processes that ligands produced, computational techniques have been successfully employed in the pharmaceutical and nutraceutical industries. More information on how physiologically active chemicals can bind to certain enzymes can be gleaned through molecular docking studies [32]. The ligand molecules were lupeol, beta amyrin, stigmasterol, gamma sitosterol, 9,12-octadecadienoic acid, and n-hexadecanoic acid. The energy and stability of the conformer were then minimized before docking to obtain the lowest energy and a more stable conformer. The binding of certain proteins with ligands promotes the efficiency of biological activity. The analysis of the protein interaction with the ligand is an important element for drug delivery and molecular pathways information. The docking outcome within each ligand to the receptor was evaluated using the docking energy (Kcal/mol) as well as the binding of every ligand with active domains of α-glucosidase and α-amylase. For the mushroom tyrosinase enzyme, rutin had the highest binding affinity (−8.9 kJ/mol), followed by scutellarin (−8.6 kJ/mol) and Lingstroside (−8.0 kJ/mol). The reference material (kojic acid) had a binding affinity of −5.3 kilojoules per mole. In contrast to ligands binding via traditional hydrogen bonding, those engaging via van der Waals forces and other weak intermolecular forces were discovered to have higher binding affinities. In the 2D docking data, van der Waals force interactions are substantially more prevalent than conventional hydrogen bonds between amino acids. This proved that our ligands have greater enzyme binding affinities than those previously reported. 4. Materials and Methods 4.1. Plant Collection and Extraction S. glutinosus (whole plant) were collected from Abbottabad when plants were fully grown. S. glutinosus plant was identified by Dr. Sarwer from Islamia University, Bahawalpur, Pakistan and the specimen was placed in the Department of Botany’s Herbarium. The collected whole plant (08 kg) was extracted with 80% hydro alcoholic solvent (methanol and water with (80:20)) for 7 days with occasional shaking. The filtration was performed with filter paper and the solvent evaporation was conducted under vacuum through a rotary evaporator. The extract was fractionated with different solvents from low polarity to high polarity (n-hexane, chloroform, and n-butanol). The extracts are stored at the appropriate temperature (until required for further use). Molecules 2022, 27, 6885 15 of 19 4.2. Phytochemical Analysis 4.2.1. GC-MS Analysis The equipment for GC-MS was Agilent, series 6890 and the detector was Hewlett Packard, 5973. Separations were attained by a coloumnHP-5MS column (length30 m × diameter 250 µm × thickness of film 0.25 µm). An electron ionization system with high energy electrons (70 eV) was utilized for spectroscopic detection by GC-MS. The temperature of the injector was 220 ± 0.2 ◦ C and the transfer line 240 ◦ C. The temperature of the oven was programmed from 60 ◦ C to 246 ◦ C at 3 ◦ C/min. Pure helium gas was passed as a carrier at 1.02 mL/min at 210 ◦ C. Prepared extracts, 1.0 µL diluted with methanol as a solvent, were injected at 250 ◦ C in a split less method. The early temperature was positioned at 50–150 ◦ C with a rising rate of 3 ◦ C/min and held for 10 min. Finally, the temperature was amplified to 300 ◦ C at a rate of 10 ◦ C/min [33]. Detection was completed using a full scan mode between 35 to 600 m/z and with a gain factor of 5. The NIST 2011, Library was used for bioactive compounds identification. 4.2.2. LC-ESI-MS Analysis Crude methanol extracts showing significantly increased antioxidant activities were also investigated using LC-ESI-MS-MS of model LTQ XL (Thermo Scientific, Waltham, MA, USA). The electron spray ionisation direct inject method was used for both negative and positive mode identification. The capillary temperature was kept constant at 290 ◦ C. The voltage applied to the capillary was 4.7 kilovolts. We kept the sample flow rate constant at 7.8 µL/min. The 50–2000 m/z mass range was successfully controlled. The nature of the parent molecular ion dictated the energy range (5–30) over which collisions were induced for fragmentation during MS/MS. All of the samples came from the same place and under the same conditions. The ESI-MS/MS data were analysed by hand using specialised software (Xcalibur 2.0.7). The structure was elucidated using ChemDraw Ultra 12.0, and the results were correlated with previously published studies [34]. 4.3. Antioxidant Activity 4.3.1. Radical Scavenging Activity The DPPH and ABTS tests were used to assess the radical scavenging ability for extracts in accordance with the previously described procedure [35]. For the DPPH assay, whole-plant extract of 4 fractions each of 0.5 mL was added to 4 mL of DPPH (0.267 mM). The absorbance of different extracts was calculated at mg 517 nm. Trolox equivalents per gram of dry extract (mg TE/g extract) were used to calculate the findings. The ABTS assay was carried out by incubating the 2 mL of ABTS solution (2.5 mM), 0.5 mL of each extract solution, and 2.45 mM potassium persulfate (equal volume) for 30 min in the dark and at 734 nm absorbance was measured. Trolox equivalents per gram of dry extract (mg TE/g extract) were used to calculate the findings. 4.3.2. Reducing Power Assays CUPRAC and FRAP assays have been used to determine the reductive potential of S. glutinosis whole-plant extracts, in accordance with previously described procedures [36]. For cupric ion reducing activity (CUPRAC assay), each extract (0.5 mL) was mixed with 10 mM CuCl2 (1 mL), 7.5 (mM) neocuproine (1 mL), and 1M NH4 Ac buffer at pH 7.0 (1 mL). The absorbance was estimated at 450 nm after 30 min of incubation at room temperature. In the same manner, the blank sample was also prepared, other than the extract. The measurement unit was milligrams of Trolox equivalents per gram of dry extract (mg TE/g extract). For ferric-reducing antioxidant power (FRAP), 0.5 mL of extract solution in methanol (10 mg/10 mL) was vortexed with 2 mL of a FRAP reagent, and 225 µL of water was added and warmed at 37 ◦ C. Then, the absorbance was read at 593 nm. The FRAP values were measured as milligrams of Trolox equivalents per gram of dry extract (mg TE/g extract). Molecules 2022, 27, 6885 16 of 19 4.3.3. Total Antioxidant Activity The total antioxidant capacity of the extracts obtained from S. glutinosus was evaluated by using the phosphomolybdenum method in agreement with the previously described procedure [37], with few modifications. First, 0.5 mL of extract solutions with methanol (1 mg/1 mL) was added to reagent mixture consisting 0.6 M sulfuric acid (0.6 M), sodium phosphate (28 mM), and ammonium molybdate (4 mM). The mixture was incubated for 90 min at 95 ◦ C and absorbances were recorded at 695 nm beside a blank sample having 0.5 mL methanol with a 3 mL reagent mixture. The measurement unit was milligrams of Trolox per gram of dry extract. 4.3.4. Metal Chelating Activity The metal chelating assay was performed in accordance with the previously described procedure [17], with some modifications. The fraction solution with methanol (0.5 mL) was added to 0.05 mL FeCl2 (2 mM). The reaction was in progress, using 0.2 mL ferrozine (5 mM). Likewise, a blank sample was prepared without ferrozine. The absorbances of all fractions were recorded after incubation at room temperature for 10 min at 562 nm. The milligrams of EDTA equivalents per gram of dry extract (mg EDTAE/g extract) were used for measurement. 4.4. Enzyme Inhibitory Activities The capacity of the different extract/fractions obtained from S. glutinosus to inhibit the α-amylase and α-glucosidase was described by the previously outlined procedure [17]. For α-amylase inhibition assay, the reaction mixture containing the different fractions of extract solution (0.5 mL) and alpha-amylase solution (10 µ/mL, 1 mL) with phosphate buffer (6 mM sodium chloride (pH 6.9) was put into the starch solution (0.05%, 0.5 mL). HCl (0.5 mL, 1 M) and 1 mL of iodine-potassium iodide solution were added to stop the reaction. The reaction mixtures were incubated for 10 min at 37 ◦ C. The blank was prepared with the same procedure without the extract. The absorbance readings were noted at 630 nm. Milligrams of acarbose equivalents per gram of dry extract were the measuring unit. The α-glucosidase inhibition assay was followed with the addition of 0.5 mL of different fraction solutions in equivalent concentrations of 0.5 mL glutathione (0.5 mg/mL) with α-glucosidase solution (0.2 u/mL) in phosphate buffer (pH 6.8) and PNPG (10 mM). After 15 min, the reaction was stopped with the addition of 0.5 mL of sodium carbonate solution (0.2 M). The absorbance readings were noted at 400 nm. The results were expressed in milligrams of acarbose equivalents per gram of dry extract (ACAEs/g extract). For tyrosinase inhibition, plant extracts were tested for their ability to inhibit the enzyme by the standard method already reported [38]. Kojic acid equivalents (KAE/g) were used to quantify the inhibitory effects on tyrosinase enzyme. 4.5. In Silico Analysis Computer-aided molecular modeling was used to examine the conformational relationship between the compound and enzyme. The drug database was used for downloading the 3D structure of ligand molecules. Crystal structures of α-glucosidase (3F5L), α- amylase (3BC9), and tyrosinase (5M6B) were downloaded from the RCSB PDB protein data bank http://www.rcsb.org/pdb (accessed on 5 January 2022). The structure file (XML and PDB format) was converted to PDBQT format using Open Babel 2.4.1. AutoDock Vina, which was offered by the server, was used to determine the required hydrogen atoms. Auto grid software with connected grid data was employed for blind docking. The initial position, orientation, and torsions were all randomized (Santos et al., 2016). The energy and stability of the conformer were then minimized before docking to obtain the lowest energy and a more stable conformer. The extracted data were compared and validated with the experimental data for α-glucosidase and α-amylase complexed with acarbose ligand [39]. According to a recent study, most of the protein–ligand bindings are based on hydrogen Molecules 2022, 27, 6885 17 of 19 bonds, ionic interactions, and van der Waals interactions. As a result, they were specifically targeted in this work by using Biovia/Discovery Studio 2021. 4.6. Statistical Analysis The average of three similar experiments was used to calculate the effects, which were represented through the average ± SD of value. The results were analyzed using one-way ANOVA from SPSS v. 17.0. Statical significance was considered as the value of p < 0.05. 5. Conclusions The present research has compared the biological properties and chemical characterization of different polarity solvent extract/fractions of S. glutinosus. The GC-MS analysis of chloroform and n-butanol fractions was performed and compared to provide more detail about the chemical profile. Fatty acids, phenols, monoterpenes, diterpenes, and sesquiterpenoids were identified as the key classes. In terms of inhibitory effects and in silico studies towards tyrosinase, α-amylase, and α-glucosidase, all extracts demonstrated different capabilities against these enzymes, and in silico studies of six selected compounds from GC-MS also provide the basis for ant-diabetic potential. Furthermore, molecular modelling of three flavonoids identified through LC-ESI-MS were docked to the tyrosinase enzyme to validate the plant’s tyrosinase inhibition potential. Based on our observation, S. glutinosus could be recognized as a promising potent biological agent possessing antioxidant, anti-diabetic, and anti-melanogenic properties. The high number of flavonoids and phenols identified in LC-ESI-MS analysis of the plant S. glutinosus in the present study may account for its powerful antioxidant and enzyme inhibition potential. However, further research concerning isolation, identification, and description of its bioactive compounds is essential to discover its potential applications in the field of medicine. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27206885/s1, Table S1: List of abbreviations; Table S2: Pharmacological properties of various species of the genus Strobilanthes [40–50]. Author Contributions: Conceptualization, M.A., U.K. and H.S.; methodology, S.A., U.K., G.A.M.M. and M.A.; software, N.J., U.K. and M.M.I.; validation, M.A.A., I.P. and J.B.; formal analysis, H.S.; investigation, U.K.; resources, S.A. and Z.M.E.-B.; data curation, S.K. and A.H.L.; writing—original draft preparation, M.A., U.K. and H.S.; writing—review and editing, U.K., F.S.A., A.A.S.A. and H.S.; project administration N.J., A.H.L. and I.P.; funding acquisition G.A.M.M., M.M.I. and Z.M.E.-B. All authors have read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support of Taif University Researchers Supporting Project number (TURSP-2020/14), Taif University, Taif, Saudi Arabia. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 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