The nano mixture of ethanol extract ketumbar (Coriandrum sativum L.) seed and putri malu (Mimosa pudica L.) leaves potentially reduced blood glucose levels in hyperglycemia wistar rat

The formation of nano extracts can increase the bioactivity of a compound in the body. The purpose of this study was to determine the ability of a nano mixture of Coriandrum sativum L seed and ethanol extract of Mimosa pudica L leaves to reduce glucose levels in hyperglycemic rats, compared to an ethanolic extract mixture of Coriandrum sativum L seeds and Mimosa pudica L leaves. The size and zeta potential characterization of the ethanolic extract mixture of Coriandrum sativum L seed and Mimosa pudica L leaves was carried out using a Particle Size Analyzer (PSA). The results of characterization with PSA showed that the nanoparticle size of the ethanolic mixture of Coriandrum sativum L seeds and Mimosa pudica L leaves was 184.4 nm and the zeta potential value was -1.62 mV. The particle size obtained is in accordance with the herbal nano size, which is 50-300 nm. Identification of compounds in the ethanolic extract mixture of Coriandrum sativum L seed and Mimosa pudica L leaves by LC-MS/MS, found 7 compounds suspected of antihyperglycemia, namely phenylalanine, indole, 4-methoxy benzaldehyde, 2,5-dimethoxy benzaldehyde, apigenin, 2-flouren-9-ylidenemethyl-pyridine, and 5-Pentyl-1,3-benzenediol. The activity test results showed that the nano ethanol extract mixture of Coriandrum sativum L seed and Mimosa pudica L leaves was more effective in lowering blood glucose levels (79.29%) compared to the ethanolic extract mixture of Coriandrum sativum L seed and Mimosa pudica L leaves (50.88%).


Introduction
Diabetes mellitus is an ongoing condition relating to the metabolism of carbohydrates (specifically glucose metabolism within the body) exhibiting the manifestation of hyperglycemia.It is distinguished by consistently heightened levels of blood sugar and is subject to fluctuations caused by irregularities in the secretion of insulin, its action, or a combination of both.Elevated blood glucose concentrations induce oxidative stress, arising from an imbalance between the abundance of free radicals and the production of endogenous antioxidants [1].There are many types of diverse free radicals, with the prevailing ones present in the biological systems of the organism being reactive oxygen species (ROS).These reactive oxygen species (ROS) are generated through the homolytic cleavage of a covalent bond within a molecule or the unpaired electron of an atom [2].
Indonesian exhibits remarkable biodiversity, harboring a plethora of antioxidant compounds.This natural abundance has been harnessed and passed down through successive generations, manifesting in the consumption of various plant-based sources, including vegetables and fruits.These botanical entities are replete with an abundance of phenolic and flavonoid compounds, which bestow upon them a formidable antioxidant prowess.Flavonoid compounds possess the ability to sequester reactive oxygen species (ROS), impede the activity of ROS-generating enzymes, and engage in reactions with non-mutated cells, including lipid peroxidation and deoxyribonucleic acid (DNA) impairment, thereby averting oxidative stress [3].
Flavonoids are thought to generate damaged pancreas cells, increase insulin secretion, and increase antioxidant enzyme activity [4].Flavonoids help antioxidant enzyme control blood sugar and prevent the complication of hyperglycemia through free radical scavenging, and reduction of oxidative stress [5].
Marella managed to collect several pieces of literature and list 418 species from 133 plant families that have antidiabetic activity, two of which are C. sativum and M. pudica L. One of the active compounds discovered in C. sativum is apigenin, while M. pudica contains phenylalanine.These compounds are widely distributed in the natural world and are present in significant amounts in various botanical specimens and fruits.Additionally, they exhibit hypoglycemic properties [6].
The ethanolic extract of C. sativum seeds and M. pudica leaves was identified using Liquid chromatography Mass Spectrometry (LC-MS/MS).The results of identification revealed a number of chromatographic peaks that had different retention times.The end result is a chromatogram, and the mass spectra of each peak are then examined using the masslynx program to interpret the chromatogram and identify the mass of each compound.Based on the similarities between the spectra and the database, the masslynx application may also assign the substance disclosed in the mass spectra.The obtained molecular formula was then confirmed on the website www.chemspider.comfor the compound's name and structure to determine its predicted compounds.It was thought that the ethanol extract of coriander seeds contained three active compounds: apigenin, 2-flouren-9ylidenemethyl-pyridine, and phentyl-1,3 benzenediol, which is an anti-diabetic.In the meanwhile, it was believed that M. pudica leaves contained active substances, such as apigenin, and luteolin as antidiabetic and antidiuretic.
Given that the active components have undergone dissolution, entanglement, and encapsulation, it follows that nanomaterials, characterized by their solid colloidal particles comprising macromolecular substances, can serve as a proficient medium for drug delivery [7].Drugs diffuse more readily in the blood and take effect more quickly when they are delivered via nanosystems [8].The chitosan polymer, a nanomaterial, is commonly utilized as a secure pharmaceutical delivery system for human subjects [9].The chitosan, upon undergoing tripolyphosphate-ionic crosslinking, exhibited a modest degree of swelling.It is also facile to manipulate the cross-linked chitosan to exhibit the desired attributes, such as crystallinity, density, and hydrophilicity [10].The ionic gelation technique, employing chitosan and sodium tripolyphosphate, was additionally employed to successfully synthesize the chloroform nano fraction derived from Kaempferia rotunda.The particle size analysis yields a range of 184 to 877 nm, while the zeta potential value ranges from +1.62 mV to 1.83 mV [9].
Based on the aforementioned description, the investigator performed an analysis/testing of nano chitosan-tripolyphosphate-C.sativum seed and M. pudica ethanol extract in their potential to reduce blood sugar levels due to their antioxidative properties, which involve the sequestration of reactive oxygen species (ROS), inhibition of ROS-generating enzymes, preservation of lipophilic antioxidants, and promotion of enzymatic antioxidant activity.
Flavonoids exhibit diverse biological functionalities, however constrained by their restricted bioavailability stemming from suboptimal absorption within the gastrointestinal tract and extensive biotransformation within the intestinal milieu.In conjunction with the rapid excretion of flavonoids from the system, augmenting the bioavailability of flavonoid composition shall yield health benefits through enhanced intestinal absorption capacity, ameliorated transportation mechanism, heightened metabolic resilience, and the alteration of absorption location from the large intestine to the small intestine.This can be achieved by employing nanotechnology to create a blend comprising nanochitosan-tripolyphosphate, ethanol extract derived from the seeds of C. sativum, and leaves of M. pudica.The nano formulation comprising of chitosan-tripolyphosphate, ethanol extract from C. sativum seeds, and ethanol extract from M. pudica leaves would enhance the solubility and bioavailability of bioactive compounds, thereby facilitating a favorable and more enduring effect at reduced doses.The in vivo confirmation of the nano-chitosan-tripolyphosphate-C.sativum L and M. pudica extract ethanol was conducted using experimental animals, specifically hyperglycemic Wistar rats.The objective was to detect the effects of this combination on reducing blood sugar levels.
Ionic gelatin technique for producing nano chitosan tripolyphosphate C. sativum L and M. pudica extracts employing chitosan as a nano extract framework and tripolyphosphate as a stabilizer.This research will result in nanotechnology.The nano herbal chitosan-tripolyphosphate C. sativum L and M. pudica were used to reduce blood sugar levels in streptozotocin-induced hyperglycemic Wistar rats.The physical chemistry characteristics of nano extracts of C. sativum and M. pudica were characterized using zeta potential and PSA (Particular size analyzer).

Materials
C. sativum and M. pudica taken from market Kumbasari Denpasar and Gianyar Bali-Indonesia.They were determined at the UPT Plant Conservation Center of the Eka Karya Botanical Gardens, Bali, Indonesian Institute of Sciences.The following chemical was used 96% ethanol, distilled water, streptozotocin (Sigma Aldrich), chitosan, and tripolyphosphate, Filter paper was also utilized.Male Wistar rats aged 2-3 months and weighing 150-200 g were the experimental animals examined in this study.

Ethanol extract of Coriandrum Sativum L and Mimosa Pudica L
C. sativum and M. pudica powder samples were immersed with 96% ethanol for 48 hours (maceration), the solvent was then evaporated at 45 degrees Celsius.The moisture content was then calculated (7.55%).It is a necessity that the water content of the extract not exceed 10% when used in herbal medicine.This Extract was used for preparing nano chitosan-tripolyphosphate-extract ethanol of C.sativum and M.pudica mixture.

Ethanol extract of Coriandrum Sativum L and Mimosa
Pudica L 100 mL of distilled water was added to the thick ethanol extract of C. sativum seed and M. pudica leaves, which weighed 2 grams, had been dissolved in 50 mL of 96% ethanol. 1 gram of tripolyphosphate and 1 gram of chitosan were each dissolved in 100 mL of 1% glacial acetate and distilled water, respectively.After that, a magnetic stirrer was used to combine and swirl the three solutions for around two hours.chitosan-tripolyphosphate-mixture extract coriander seed and mimosa leaves nanoparticles were separated by centrifugation.After being separated, the resulting particles were placed in the freezer (-4 o C) for 2 days.Then storage is moved to a refrigerator ( 300 o C) [11].

Ethanol extract of Coriandrum Sativum L and Mimosa Pudica L
Zeta potential measurement of C. sativum seed and M. pudica leaves nanoparticles mixture was done by filling the cuvette with the sample until there were no more bubbles.The inlet is then covered to reduce vibration and ensure that the outcomes are unaffected.The cell penetration is positioned in front of the cell support's arrow.Additionally, the electrode cell is examined to make sure that it is attached to the instrument electrode and that the tool cover is in place."Measurement start" by pushing the button.The cell was removed and then sanitized when the measurements were finished [11].

Blood glucose level
The completely randomized design of true experimental was carried out with Posttest Only Control Grup Design [12].Three groups of 27 male Wistar rats, each aged 3 months and weighing between 150 and 200 grams, were adapted for one week of laboratory acclimation.K0: Positive control group accepted 40 mg/kgBW of streptozotocin only.P2: Given streptozotocin 40 mg/kgBW and treated with ethanol extract mixture of C.sativum seed and M.Pudica leaves 50 mg/kgBW.P3: The third treatment group received nanoparticles of chitosan-tripolyphosphate-ethanol extract mixture of C. sativum seed and M.pudica leaves 50 mg/kgBW and streptozotocin 40 mg/kgBW.Blood sugar level reduction is determined by measuring it in mg/dL using the GLUCO test kit method on rats' tail vein blood on the fourth day following streptozotocin induction and the 15 th day following treatment of P2 and P3.[13].SPSS 23 Windows was used to statistically examine the blood glucose levels using ANAVA.If the outcome was significant, further a post hoc test with LSD was conducted.

Identification of active compound through LCMS/MS
The active compound of the ethanol extract of C. sativum and M.pudica were identified through LCMS/MS by comparing the spectrum of standard compounds in the database A chromatogram was obtained and examined using Masslynx V4 to determine the mass dan molecular formula of the compounds.(w/w).According to the notion of like dissolves like, polar compounds dissolve in polar solvents and non-polar compounds dissolve in non-polar solvents [15], the type of extraction solvent affects the amount of bioactive molecules that may be extracted from the sample.

Decrease in blood glucose levels
The glucose concentration in the blood characteristics in Wistar rats, specifically the blood glucose levels following streptozotocin induction on day 3 and subsequent administration (15 days) of a single dose of ethanol extract, is depicted in Figure 1.On the 15 th day, nano chitosan-tripolyphosphate-C.sativum and M. pudica ethanol extract were injected.The rats subjected to the administration of the ethanol extract combination of C. sativum and M. pudica at a dose of 50 mg per kilogram of body weight exhibited an average blood glucose concentration of 323.7 milligrams per deciliter in the untreated group K0 (solely induced by streptozotocin).A group of rats fed C. sativum seed and M. pudica leaves ethanol extract nanoparticles at a dosage of 50 mg/kgBW had an average blood glucose level of 136.33 mg/dL.The average blood glucose level in Group P2 was 99.4 mg/dL.When blood glucose levels in rats are measured, the mixture of C. sativum and M. pudica leaves ethanol extract nanoparticles at 50 mg/kgBW is more efficient.The reduction in blood glucose levels in Wistar rats is postulated to be attributed to the presence of secondary metabolites with antihyperglycemic and antioxidant properties.Hyperglycemia induces a surge in oxidative stress, concomitant with a decline in endogenous antioxidants, which are the antioxidants created within the human body.The intake of natural antioxidants serves as a protective measure against the onset of diabetes by inhibiting the peroxidation process that inflicts destruction upon pancreatic beta cells.C. sativum seeds, according to Yulianty [24], have various active substances that help reduce blood glucose levels.The presence of polyphenolic, alkaloids, and flavonoids was discovered using phytochemical screening [25].
Utilizing a micro syringe, a specimen of ethanol extract from C. sativum, obtained through solidphase extraction (SPE) employing a methanol eluent, was introduced into the LCMS/MS instrument utilizing a stationary phase/C18 column (Octadecyl silane) with a maximum injection volume of 5 µL.The chromatographic data was visualized in the form of a chromatogram, subsequently subjected to analysis using MassLynx v 4.1 software in order to ascertain the molecular mass of the compound obtained during each retention time.On the website www.chesmspider.com,the structure and nomenclature of the compound were verified.Because it is separated by firing one proton (H + ) during the separation in MS, one H atom was subtracted.The chromatogram data are shown in Figure 2. The substances were identified in good agreement with the data available on the mass bank and ChemSpider based on the results of the study on the apps http://www.massbank.ip/and http://chemspider.com.According to the database, the presence of phenylalanine, indole, 4-methoxy benzaldehyde, 2,5-dimethoxy benzaldehyde, apigenin, 2-flouren-9-ylidenemethyl-pyridine, and 5-Pentyl-1,3-benzenediol was found in C. sativum seed ethanol extract.
Apigenin, a flavonoid derivative compound, manifests prolifically in the natural realm and occurs in notable quantities within a diverse array of botanical specimens and fruit entities, encompassing celery, onions, chamomile flowers, oranges, and tea leaves.It exhibits anticancer, anti-inflammatory, and antioxidant characteristics while maintaining non-toxic attributes.Flavonoids, which are chemical compounds with antioxidant properties, have been found to play a significant role in the management of diabetes [6].The apigenin molecule, derived from flavonoids, was hypothesized to possess antihyperglycemic properties in this investigation.

Mechanism of flavonoid release
Flavonoids exhibit antioxidant properties by effectively neutralizing free radicals.The flavonoid antioxidant activity is predominantly dictated by the structure of their molecules, particularly the composition of substituents present within their molecular framework [30].The flavonoids entrapped within the carrier matrix exhibit inhibitory effects on degradation [31], and the liberation of flavonoids from nano-chitosan-tripolyphosphate-C.sativum seed and Mimosa pudica leaves extract ethanol, which is affixed to the surface/adsorbed, transpires via flavonoid decomposition or desorption facilitated by diffusion and erosion [19].The immiscible polymer matrix hinders the dispersion of flavonoid molecules in an aqueous medium.This process happens in systems with a diffusion barrier made of hydrogel crosslink polymers.The diffusion barrier decreases as the hydrogel swells, and vacancies occur in the gel structure [32].The release of flavonoids occurs upon the absorption of the buffer liquid by the nano-chitosan-tripolyphosphate-C.sativum and Mimosa pudica extract, resulting in the expansion of the gel.The observed phenomenon of swelling results in an elevation of the fluid within the nano-chitosanripolyphosphate-C.sativum and Mimosa pudica L extract ethanol.Additionally, it leads to an expansion of the pores and dimensions of the nano-chitosan-tripolyphosphate-C.sativum extract ethanol.Consequently, this facilitates the permeation of flavonoids into the adjacent environment [9].
During the interim, the phenomenon of flavonoid liberation via attrition can be attributed to the gradual degradation of the protective coating layer.The nano chitosan-tripolyphosphate-C.sativum and Mimosa pudica L extract ethanol corroded as a result of a hydrolysis reaction that liberated flavonoids from the coating system [19].Chitosan, a type of polysaccharide polymer, exhibits the ability to undergo expansion and gel formation upon exposure to acidic conditions [33].The gel exhibits a semisolid state comprising of two interpenetrating continuous phases, specifically the solid phase consisting of an asymmetric solid phase, which subsequently occupies the interstitial space [20].Chitosan polymers, characterized by their extended molecular structures, undergo intermolecular crosslinking with tripolyphosphate, resulting in a water-capturing appearance [22].The gel sheath surrounding the nanochitosa-tripophosphate-C.sativum and M. pudica ethanol extract, which is viscous and can spread over several hours, is a natural barrier for flavonoid to be released, thereby aiding in the retention of flavonoids in their dosage until all flavonoids have been released.The gel form will be maintained for several hours, delaying flavonoid release for an extended period [34].Liquid movement causes the long structure of chitosan polymer chains to open and intermolecular bonds to break.This causes the hydrogel layer on the surface of the nano chitosan-tripolyphosphate-C.sativum and M. pudica extract to be progressively loaded, allowing the flavonoids contained within the hydrogel layer to be released [35].

Characterization with particle size analyzer (PSA)
A particle size analyzer (PSA) is used to calculate the zeta potential value and particle size of a basic substance [10].Table 1 reveals that the zeta potential value of nano chitosan-tripolyphosphate-C.sativum and M. pudica extract ethanol is -1.62 mV, indicating that it has the capacity as an introduction to bioactive material [32].Nano chitosan-tripolyphosphate has the smallest particle size, which is 119.3 nm, indicating that it has the most surface area of the three samples [19].

Conclusion and recommendations
Nano extracts are made by encapsulating bioactive substances from a mixture C. sativum and M. pudica ethanol extract with chitosan and tripolyphosphate polymer as material for encapsulation framework for the development of delivery systems for bioactive substances in the medical field extensively.
Recommendations for further research include: 1) Nano extract C. sativum and M. pudica are expected to be utilized as a nano herbal for hyperglycemia therapy through the free radical pathway by observing glucose biomarkers.
2) The encapsulation of nano C. sativum and M. pudica extract into nano chitosan tripolyphosphate by crosslinking with the ionic gelation method causes changes in physical-chemical properties such as compound functional groups that carry the characteristics of zeta potential bioactive substances as particle size.3) Giving of nano extract of C. sativum and M. pudica causes glucose blood, and MDA to decrease using streptozotocin-induced Wistar rats (rats to become hyperglycemia).4) Conducted research on the release rate of bioactive substances encapsulated in nano chitosantripolyphosphate-C.sativum and M. pudica ethanol extract.5) Continuing research by clinicians to conduct clinical trials regarding the activation of antihyperglycemic nano chitosan-tripolyphosphate-C.sativum and M. pudica extract ethanol in humans.

3 . 1 .
Coriandrum Sativum L and Mimosa Pudica L extract Following a 48-hour maceration of 2000 grams of powdered Coriandrum sativum L and M.pudica with ethanol solvent, 147.25 g of thick blackish-dark yellow ethanol extract with a yield percentage of 7.55% was achieved.Because the proportion of water contained in C. sativum L and Mimosa pudica L powder fulfilled the simplicia standard, ethanol selected as the solvent.Excess water content may promote easy microbial development in the simplicia, resulting in the hydrolysis of active chemicals and a reduction in the effectiveness of the extraction solvent [14].The samples of C. sativum and M.pudica were macerated in 96% ethanol.The ethanol extract of C. sativum L and M. pudica yielded 14.74%

Figure 2 .
Figure 2. Chromatogram of liquid chromatography with tandem masspectrometry ethanol extract mixture of C. sativum seed and M.pudica leaves

Table 1 .
Zeta potential and particle size