Department Pharmacognosy-Phytochemistry, Faculty of Pharmacy, Universitas Muslim Indonesia, Makassar-South Celebes, Indonesia

ABSTRACT

Diabetes is one of the metabolic diseases indicated by hyperglycemia resulting from production in insulin secretion, insulin action, or both. Type 2 diabetes, which accounts is ~90–95% of those with diabetes, the cause is a combination of resistance to insulin action and an inadequate compensatory insulin secretory response. Adequate glycemic control is thus one of the key factors to treat and/or reduce the diabetes and many plants have been used to reduce the glucose level by inhibiting the α-glucosidase that breaks down starch and oligosaccharide to glucose. Acorus calamus L (AC) had been used in the folk medicine to treat diabetes. In vitro α-glucosidase assay was carried out by measuring the release of p-nitro phenol, the insulin sensitizing activity, AC altogether brought down fasting serum glucose, and smothered the rebellion of blood glucose levels after 2g/kg glucose stacking in ordinary mice, in silico study showed that chemical compounds on AC can inhibit α-glucosidase and the later investigate is demonstrated to decide the impacts and atomic instruments of AC on glucagon-like peptide-1 (GLP-1) expression and discharge related to its hypoglycemic impacts.

Keywords: Acorus calamus L, folk medicine, α-glucosidase, in silico, molecular mechanism, type 2 diabetes.

Diabetes mellitus could be a illnesses of disarranged digestion system, influenced by a combination of innate and natural components, coming about in hyperglycemia due to abandons in either affront discharge or affront activity within the body. Constant hyperglycemia amid diabetes causes glycation of body proteins that in turn leads to secondary complications affecting eyes, kidneys, nerves and courses¹. Diabetes mellitus (DM) could be a common endocrine framework malady that causes metabolic disarranges and which leads to numerous organ harm disorder. Clinical chief naval officer diabetes is partitioned into two sorts, with more than 90% of patients having Sort II diabetes². The number of diabetes cases was 171 million in 2000 and is anticipated to rise to 366 million in 2030³. Acting as a key chemical for carbohydrate assimilation, intestinal α-glucosidase could be a glucosidase found at the epithelium of the little digestive tract. α- glucosidase has been recognized as a helpful target for the balance of postprandial hyperglycemia, which is the earliest metabolic abnormality that occurs in Type 2 DM⁴.

Type 2 diabetes is the for the most part type of diabetes, representing around 90–95% of every diabetic case. This type of diabetes typically starts with insulin lack of care, a condition where muscle, liver and fat cells don't react to insulin appropriately. The pancreas in the end loses the capacity to deliver and discharge enough insulin in light of food consumption. Gestational diabetes is brought about by hormonal changes during pregnancy or by insulin inadequacy. The glucose neglects to enter cells, in this manner expanding the glucose level in the blood. High blood glucose, otherwise called hyperglycemia, can harm nerves and veins, prompting Complex illnesses, for example, coronary illness, stroke, kidney brokenness, visual impairment, nerve issues, gum contaminations and removal⁵. Type 2 diabetes mellitus could be a complex, multi factorial infection. Oxidative stretch has been recommended to be a contributory calculate in advancement and complication of diabetes⁶^,⁷. In later a long time, characteristic cancer prevention agents are utilized in dietary, pharmaceutical and corrective to supplant engineered cancer prevention agents⁸. Inquire comes about finding that a few antioxidant substances separated and distinguished from restorative plants had great comes about on auto oxidation in vitro and in vivo⁹^,¹⁰. Postprandial hyperglycemia is the foremost vital wellbeing issue within the 21^st century. α-Glucosidase inhibitors decreases postprandial blood glucose level. Looking for strong normal substances as glycosidase inhibitors is exceptionally vital to remedy diabetes¹¹.

The blood glucose levels of a sound man are 80 mg/dL on fasting and up to 160 mg/dL in the postprandial state. Diabetes mellitus is described by intermittent or steady hyperglycemia, and is analyzed by exhibiting one of the accompanying: Fasting blood glucose level at or upper 126 mg/dL or 7.0 mmol/l. plasma glucose at or upper 200 mg/dL or 11.1 mmol/L two hours after a 75 g oral glucose load in a glucose resistance test. Plasma glucose at or over 200 mg/dL or 11.1 mmol/l. Two fasting glucose estimations over 126 mg/dL or 7.0 mmol/l or arbitrary glucose level >200mg/dL on two events is viewed as indicative for diabetes mellitus. Patients with fasting sugars somewhere in the range of 6.1 and 7.0 mmol/L (110 and 125 mg/dL) are considered to have debilitated fasting glucose and patients with plasma glucose at or over 140 mg/dL or 7.8 mmol/L two hours after a 75 g oral glucose load is considered to have weakened glucose resilience¹².

After over than ten a long time of inquire about unraveling complex metabolic control systems, solutions able of a secure inversion of sort 2 diabetes are still not accessible. Verifiably, complex illnesses have over and over demonstrated to be insubordinate to the finest mono-therapeutic approaches¹³. A few illustrations of combination treatments have generally overcome such challenges, outstandingly for the treatment of extreme hypertension and tuberculosis. Corpulence and its results, such as sort 2 diabetes, have demonstrated to be similarly safe to helpful approaches based on single solutions. Suitable administration of sort 2 diabetes regularly requires adjunctive medications, and the later enrollment of some compound blends has set the point of reference for combinatorial treatment of weight. On the other hand, twofold or triple helpful combinations are more troublesome to development to administrative endorsement. Taking after moved forward understanding of the atomic premise for metabolic benefits taking after bariatric surgery interventions, several classes of novel uni-molecular or independent combination therapeutics were discovered. These unused classes of medicate candidates are based on gastrointestinal hormones, offer adequacy predominant to right now endorsed alternatives and appear to have potential to completely switch human weight and sort 2 diabetes¹⁴^,¹⁵. Besides, intestine peptide-based cell-specific focused on conveyance of little atoms offers extra potential for novel metabolic exactness drugs and decreased systemic side impacts. In this introduction the revelation, pre-clinical approval and to begin with clinical tests of peptide hormone poly-agonist medicate candidates as well as of combinatorial single particle helpful candidates will be summarized, counting already unpublished perceptions¹⁶.

Western diabetic drugs adjust hypoglycemia by supplementing affront, moving forward affront affectability, expanding affront emission from the pancreas and/or glucose take-up by tissue cells. Beneath ordinary conditions, pancreatic β-cells discharge adequate affront to preserve blood glucose concentration inside a limit extend (72–126 mg/dL). The affront incitement taken after by cascade signaling improves glucose admissions, utilization and capacity in different tissues. In diabetic patients, the body loses affront creating capacity as a result of pancreatic β-cell apoptosis or affront lack of care. The cytokines, lipo-toxicity and gluco-toxicity are three major jolts for β- cell apoptosis¹⁵. The medicines of diabetes incorporate slim down, work out, utilize of verbal hypoglycemic specialists and affront are the essential shapes of treatment for diabetes. Right now accessible engineered antidiabetic operators other than being costly deliver genuine side impacts. Home grown pharmaceutical is separated from as of now accessible restorative choices. There are numerous home grown solutions have been suggested for the treatment of diabetes mellitus. Utilizing of restorative plants has the advantage of having no side impacts¹³. Conventional plant medicines have been utilized all through the world for the treatment of diabetes mellitus. History appeared that therapeutic plants have been utilized in conventional mending around the world for a long time to treat diabetes; this is often since such home grown plants have hypoglycemic properties and other advantageous properties, as detailed in logical literary works¹⁴.

Ethno pharmacological overviews demonstrate that more than 1200 plants are utilized in conventional medication for their affirmed hypoglycemic action¹⁷. Therapeutic plants, since times immemorial, have been utilized in essentially all societies as a source of pharmaceutical. A consider of old writing shows that diabetes was reasonably well known and well-conceived as a substance in old India. The information of the framework of diabetes mellitus, as the history uncovers, existed with the Indians since ancient age. Its most punctual reference (1000 BC in the Ayurveda writing) is found in legendary shape where it is said to have begun by eating Havisha²^,¹⁸.

The NAPRALERT database records over 1200 species of plants speaking to 725 genera in 183 families expanding from the marine green growth and organisms with antidiabetic movement¹⁹. Over half of these have been utilized ethno- pharmacologically in conventional medication as antidiabetics, and a few 50% of these conventional cures have been examined tentatively¹⁹. The utilization of conventional pharmaceutical and restorative plants in most creating nations, as a standardizing premise for the upkeep of great wellbeing, has been broadly watched. Moreover, an expanding dependence on the utilize of therapeutic plants within the industrialized social orders has been followed to the extraction and improvement of a few drugs and chemotherapeutics from these plants as well as from customarily utilized home grown cures². Certain herbs may lower blood glucose²⁰^,²¹; however, their test results are subject to several factors. Firstly, each herb contains thousands of components, only a few of which may be therapeutically effective²². Secondly, different parts of an herb have different ingredient profiles. Moreover, different extraction methods may yield different active ingredients. Thirdly, herbal formulae containing multiple herbs may have synergistic effects¹⁵.

In Canada, following plants are used in the treatment of diabetes by the tribal people Abies balsamea (L.) Mill. Achillea millefolium L., Acorus calamus L., Aralia nudicaulis L., Aralia racemose L., Arisaema triphyllum (L.), Asarum canadense var. acuminatum Ashe, Celastrus scandens L., Cornus stolonifera Michx., Corylus cornuta Marsh., Dirca palustris L., Gaultheria procumbens L., Heracleum lanatum Michx., Juniperus communis L., Juniperus virginiana L., Kalmia angustifolia L., Ledum groenlandicum Oeder., Nuphar variegatum Durand, Picea glauca (Moench) Voss., Picea mariana (Mill.), Populus balsamifera L., Populus tremuloides Michx., Prunus serotina Ehrh., Quercus alba L., Quercus rubra L., Rhus hirta f. typhina (L.), Sassafras albidum (Nutt.) Ness., Smilacina racemosa (L.) Desf²³^,²⁴.

Study Consider from the Rhizomes of Acorus calamus L. is broadly utilized within the treatment of diabetes in conventional people pharmaceutical of America²⁵ and it wins in Merak, Banten, Indonesia to progress diabetes. In any case, the diabetic effects of Acorus calamus L. have not been completely studied yet²⁶.

Acorus calamus L (AC), also known as ‘Vacha or Sweet flag’, it has been an critical herb within the Ayurvedic medication and inborn therapeutic framework for over 100 a long time. AC rhizomes have been utilized as a single sedate or as a component of certain compound sedate arrangements within the Indian Ayurvedic framework of medication for psychoneurosis, a sleeping disorder, mania, epilepsy and loss of memory²⁷^,²⁸^,²⁹. It is additionally utilized within the treatment of hack, fever, bronchitis, irritation, sadness and other mental clutters, tumors, hemorrhoids, skin maladies, deadness and common debility³⁰, stimulant, emetic, carminative, stomachic, as cures for a few harming²⁹. AC can be found developing in Central Asia or India, Central Europe and North America. In India it is common in ranges that encompass the Himalayas. Indian AC from the Jammu zone is triploid and tetraploid; and European as well as American assortment of the AC is diploid²⁸^,²⁹^,³¹^.

Synonyms: Sanskrit: Vacha, Sadgrantha; English: Calamus, Sweet Flag; Marathi: Vekhand; Hindi: Bach, Gorbach; Tamil: Vashambu; Telgu: Vadaja, Vasa; Bengal: Bach.

Botanical description: Calamus could be a semi sea-going herb and is broadly disseminated by the edges of lakes and moderate streaming waterways, growing in shallow water or in a really damp loamy soil. It lean towards a pH within the run 5.5 to 7.5. It is perennial herb; the rhizomes commonly occur in pieces about 5 to 15 cm in length and 1 to 2 cm in thickness. They are covered with a thin brownish epidermis and cork and are much shrunken, bearing brief longitudinal wrinkle. They are marked on the upper surface with large triangular leaf scare that encircle the rhizome, springing from each side alternately; from these scars fibrous leaf trace bundle frequently project. The under surface bears an irregular zigzag line of small raised root scars that are circular and exhibit a central stele surrounded by a narrow cortex. The rhizome breaks with a short corky fracture, and is pale brown or nearly white and spongy internally. The section shows a expansive stele isolated by a yellowish line, the endodermis from a thick cortex; various little, oval, vascular bundles are scattered thought the segment. The naturally broken rhizome has a pleasing fragrant odor. Clears out are right green having sword-shaped, based equitant, thickened in center, edges wavy. Blossoms are showed up in June and July and are yellow/green in color. The blossoms are bisexual (have both male and female organs) and are pollinated by Creepy crawlies²⁸^,²⁹^,³³.

A wide assortment of chemical constituents have been detailed from the rhizomes of AC. The oil of AC rhizomes has been analyzed by different laborers for their chemical constituents. The oil was found to contain shifting concentrations of α- asarone (1), β-asarone (2), γ-asarone (3), calamene, calamenenol, calameone (4), α -pinene (5), β -pinene (6), camphene, p-cymene, eugenyl acetate, eugenol (7), isoeugenol (8), methyl isoeugenol (9), calamol, azulene (10), eugenol methyl ether, dipentene (11), methyleugenol, asaronaldehyde (12), terpinolene (13), 1,8-cineole (14), camphor (15), α-caryophyllene (16), and hydrocarbons (Figure1) The oil too contains greasy acids such as palmitic corrosive and its ester, heptylic corrosive, an ester of butyric acid.first detailed the amalgamation of asarone from 1,2,4- trimethoxybenzene by Sharma et.al 1969³³. Fractionation from the volatile oil by gas chromatography resulted in the isolation of α-asarone and β-asarone, which are the trans- and cis-isomers, respectively, of 2,4,5-trimethoxy-l-propenylbenzene³⁴.

Wu et al., detailed that ethyl acetic acid derivation division of Acorus calamus L (Pro) but not other divisions of AC could enhance 3T3-L1 cells separation³⁵, they recognized the impacts of Pro on glucose utilization of L6 cells, which are delicate to affront. Their comes out appeared in rosiglitazone upgraded glucose utilization of L6 cells in an affront subordinate way (p<0.01 vs. vehicle with affront), though metformin lifted glucose utilization free of affront (p < 0.01 vs. vehicle either with or without affront). 12.5 and 25 g/ml of ACE brought down the glucose of culture media within the presence but not within the nonappearance of affront (p < 0.05 and p < 0.01 vs. vehicle within the nearness of affront, p > 0.05 within the nonattendance of affront), and comparable comes about were watched in rosiglitazone groups. ACE pro clearly expanded affront interceded glucose utilization in L6 skeletal muscle cells, proposing that ACE may antagonize diabetes by progressing the affront affectability³⁶.

To con?rm the insulin sensitizing effects of ACE in vivo, insulin resistant affront safe diabetic db/db mice were orally administrated for 3 weeks. As a result, the values of serum glucose within the diverse treated bunches (10 mg/kg rosiglitazone, 100 mg/kg ACE, and 5 mg/kg rosiglitazone combined with 100 mg/kg ACE) declined by 40.1%, 34.1% and 49% after 2 weeks, and by 70.1%, 54.5% and 76.1% after 3 weeks, comparing with vehicle control respectively (p < 0.001). Serum triglyceride diminished signi?cantly in all treatment bunches after 1–3 weeks’ organization comparing with vehicle control. After 3 weeks’ organization, 100 mg/kg Pro appeared no signi?cantly in?uence on serum add up to cholesterol (p > 0.05), whereas 10 mg/kg rosiglitazone diminished it after 2 and 3 weeks’ organization (p<0.05), and a combination of 100 mg/kg ACE and 5 mg/kg rosiglitazone markedly decreased add up to cholesterol after 3 weeks’ treatment (p< 0.01). These comes about show that ACE discourages not only blood sugar but moreover triglyceride in stout diabetic mice, and moves forward the lowering effect of total cholesterol caused by rosiglitazone³⁶.

Our previous research find the potency of fraction n-butanol AC extract as inhibitory agent on α- glucosidase enzyme. Sample from fraction of n-butanol AC extract with column chromatography method to separated it. We use the resin to separate fraction because it is suitable for the crude extract with high polarity (hydrophilic). The result of inhibitory assay of α-glucosidase from fraction showed that the 5th fraction was the most active with IC50 value 4.87 µg ML-1 while the other fraction has not activity³⁷. Our examination utilize a Koji extricate as control from Apergillus terreus is an particularly productive maker of auxiliary metabolites has organic exercises such as inhibitory of α-glucosidase and it features a most potential action in this manner inspected the impact on postprandial blood glucose level after a supper in mice. Triana’s research on inhibition mode Koji extract against α- glucosidase was investigated. Inhibition mode of Koji extract had a combination of non-competitive and uncompetitive inhibition³⁸. In their study inhibition mode of AC extract had a non-competitive inhibition, non-competitive inhibition of AC extract may be having different structure from the compound that has α- glucosidase inhibitory activity on competitive mode like acarbose³⁷.

S.N.	Ligand/chemical compound	Receptor α-Glukosidase	Free energy (?H)	Information
1	(-)-Cadala-1,4,9-triene	α-Glukosidase	0	(-)
2	Ac ola mone	α-Glukosidase	0	(-)
3	Acoradin	α-Glukosidase	0	(-)
4	Acoragenmacrone	α-Glukosidase	0	(-)
5	Acorenon	α-Glukosidase	0	(-)
6	Acorid acid	α-Glukosidase	-7,26053 kcal/mol	(+)
7	Acorenene	α-Glukosidase	0	(-)
8	Aristolene	α-Glukosidase	0	(-)
9	Beta-acarone	α-Glukosidase	-7,62818 kcal/mol	(+)
10	Beta - Guanine	α-Glukosidase	0	(-)
11	Calacone	α-Glukosidase	-7,65883 kcal/mol	(+)
12	Calamusenone	α-Glukosidase	0	(-)
13	Calarene	α-Glukosidase	-2,9378 kcal/mol	(+)
14	1-ethenyl-1-methyl-2,4-di(prop-1-en-2-yl)cyclohexane	α-Glukosidase	-8,04385 kcal/mol	(+)
15	Delta - cadienene	α-Glukosidase	0	(-)
16	Apihsyobunon	α-Glukosidase	-7,74775 kcal/mol	(+)
17	Isoacolamone	α-Glukosidase	0	(-)
18	Isocaespitol	α-Glukosidase	-8,28388 kcal/mol	(+)
19	Isocalame ndiol	α-Glukosidase	0	(-)
20	Isoshyobunon	α-Glukosidase	0	(-)
21	Methylsoegenol	α-Glukosidase	-7,92367 kcal/mol	(+)
22	Preisocalamendiol	α-Glukosidase	0	(-)
23	Shyobunon	α-Glukosidase	-7,75501 kcal/mol	(+)

On the next research we find that one of isolate from this fraction can inhibit the α-glucosidase with IC50 17.89 µg/mL³⁹. For the additional research we have been use HPTLC method to find out the fingerprint of AC compounds of leaf and rhizome. This research showed that β-asarone is the major compound on the leaf⁴⁰.

AC and ACE expanded affront discharge in HIT-T15 cells as that of gliclazide. As in vivo comes about, ACE (400 and 800 mg/kg) essentially diminished fasting serum glucose, and stifled the increment of blood glucose levels after 2g/kg glucose stacking in typical mice. In expansion, Pro as a mixed-type inhibitor hindered alpha-glucosidase action in vitro with an IC₅₀ of 0.41μg/ml, and 100mg/kg of it clearly diminished the increment of blood glucose levels after 5g/kg starch stacking in typical mice. Separated from its affront sensitizing impact, ACE may have hypoglycemic impacts through instruments of affront discharging and alpha-glucosidase restraint, and in this way progresses postprandial hyperglycemia and cardiovascular complications⁴¹.

The large scale atom of the protein α-glucosidase was gotten through the protein information bank with the code 1lwj within the download NCBI site. Models of chemical compounds contained in A. calamus L. gotten through the location take out from databases "jamu" Knapsack⁴² and make the 2D and 3D utilizing by Chemsketch on the freeware adaptation. At that point docking utilized Argus lab. Docking results as shown on Table 1, and Figure 3 and Figure 4. Recently, In silico has lead an important role in drug design and finding the drugs substances. In which micro molecule is virtually docked in to a drug target and the binding affinities are estimated using simplified free energy calculation method. Many programs capable of carrying out virtual screening have been developed; most of them are pay ware.

One freely available docking software package potentially capable is Argus Lab. Argus lab was originally developed as molecular modeling software. It provides users with molecular building analyses, the ability to perform various molecular calculation and molecular structure visualization capabilities⁴³. Atomic docking examination capability was included to most recent adaptation of Argus Lab (ver.4.0.1). Argus Lab can be effortlessly utilized indeed by tenderfoot in computational docking and can run utilizing windows (Microsoft Corp)⁴³.The chemical α-glucosidase is the chemical capable for the change of carbohydrates into glucose. Starch are processed by chemicals within the mouth and intestines into less complex sugars which can at that point be retained into the body and move forward blood sugar⁴⁴. AC obtained several compounds such as Beta asarone, Acoradine, Methylsoegenol, 1-ethenyl-1-methyl-2,4-di(prop-1-en-2-yl)cyclohexane, Isocaesp-itol, Acoragermacrone, Preisocalamendiol, Shyobunon, Epishyobunone, Isocalamone, Acolamone, Aristolene, (-)-Cadala-1,4,9-triene, Isocalamendiol, Calacone, β-gualene, Calamusenon, Acoronene, Acorid acid, Calarene, Acorenone through the site. Take out "jamu" Knapsack and made in the formula structures of 2D and 3D using the program ACD/Chemsketch. Then docking used Argus lab Program are visualized by Pymol program. Docking results showed activity in the compound 1- ethenyl- 1-methyl-2,4-at (prop-1-en-2-yl) Cyclohe-xane with free energy -8.04385 kcal/mol, and the compound Isocaespitol with a free energy -8.28388 kcal/mol.

Chemical component that has the lowest free energy showed the most stable affinity that is expected to have good medicinal properties as well. Docking results showed activity in the compound 1-ethenyl- 1-methyl-2,4-at (prop-1-en-2-yl) Cyclohexane with free energy - 8.04385 kcal/mol, and the compound Isocaespitol with a free energy -8.28388 kcal / mol⁴³.

ACE acts as an antidiabetic through affront sensitizing, affront discharging and alpha-glucosidase inhibitory exercises. The show consider is planned to examine the impacts and atomic instruments of ACE on glucagon-like peptide-1 (GLP-1) expression and secretion related to its hypoglycemic effects. The hypoglycemic effect of ACE (100mg/kg, i.g.) was affirmed by testing blood glucose levels or by means of verbal glucose resilience test (OGTT) in streptozotocin (STZ) actuated hyperglycemic mice, db/db diabetic mice and diet-induced hefty (DIO) mice. Plasma affront, GLP-1 levels and intestinal GLP-1 related quality expression were decided in STZ induced and db/db diabetic mice. The in vitro effects of ACE (12.5μg/ml) on the expression and secretion of GLP-1 were detected in NCI-H716 intestinal L-cells, and the relationship between ACE and particles within the signaling pathway was encouraging investigated⁴⁵. ACE (100mg/kg) altogether brought down fasting blood glucose in STZ-induced and db/db diabetic mice and progressed the OGTT in DIO mice. Affront discharging and islet defensive impacts, at the side the expanded emission of GLP-1, were watched. The expression of proglucagon quality (gcg) and post-translational processing gene prohormone convertase 3 (pc3) and the GLP-1 substance within the culture medium of L-cells eminently expanded after the ACE treatment (12.5μg/ml). At the same time, β-catenin atomic translocation happened, and its downstream protein cyclin D1 was actuated, appearing the inclusion of Wnt signaling. ACE might enact Wnt signaling to extend the quality expression of gcg and pc3 and apply incretin impacts, counting insulinotropic and islet security, to lower blood glucose levels through lifted GLP-1 discharge either straightforwardly or by implication⁴⁵.

Acorus calamus L. has been proofed as folk medicine that can cure type 2 diabetes mellitus on many mechanisms and can be used as antidiabetic related with many experiment methods.

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