Characterization and biological activity of some anti-diabetic plants: Medicago sativa Gongronema latifolium and Pterocarpus santalinoides

Plants are believed to be an essential source of new chemical substances with potential therapeutic effect. The aim of this study is to test for the GCMS analysis and FT-IR analysis of the extract of Medicago sativa , Gongronema latifolium and Pterocarpus santalinoides. FTIR spectroscopy of all selected extracts confirms the presence of alkanes, alkynes, carboxylic acids, aldehydes and nitriles functional groups while the major bioactive compounds detected in the extracts were protchaechenic acid, hydroxyl benzoic acid , b-Phenyl caffate and kaempherol.


Introduction
The hunt for natural remedies to treat illnesses has drawn a lot of interest, and the most significant source has been medicinal plants 1 .In light of the global upswing in medication resistance, toxicity, side effects, and rising costs of synthetic products, traditional medicine products have played and continue to play bigger roles in the lives of people all over the world.The synergistic and antagonistic pharmacological interaction in poly-herbal medicines provide therapeutic efficacy with little side effects.leaves and stems are abundant in vitamins, minerals, and proteins and help prevent deficiencies of certain nutrients.These plants have several medical applications, including those for treating fungal infections, restoring digestive health, and promoting breastfeeding in nursing mothers.According to several studies 2,3,4 , antioxidants are micronutrients that can stop the oxidative damage brought on by free radicals.There are numerous substances with antioxidant action found in medicinal plants.Practitioners of traditional medicine in South East Nigeria use Medicago sativa, Gongronema latifoliun and Pterocarpus santalinoides, as a folk remedy for treating diabetes mellitus.Hence we made an attempt to study the validity of these folk remedy by investigating the chemical components and functional groups in the three plants.

Plant Materials
Fresh leaves of the plants P. santalinoides, M. sativa, and G. latifolium were gotten from a farm in Umuekwune Ngorokpala L.G.A., Imo State.Dr. Mbagwu, a taxonomist from Imo State University, authenticated the leaves.The leaves were deposited into the herbarium and voucher numbers IMSUH 551, 552, and 553 were assigned to them.

Preparation of Extract
The fresh leaves of the plantswere shed, dried at room temperature of 30 0 C and dried leaves reduced to a coarse powder in a mill (Kenwood BL357).The powder (800g) was extracted with 2 L of 80% ethanol using a soxhlet extractor and concentrated in vacuo using rotary evaporator for solvent evaporation.The weight of the dried extracts were measured, and the percentage extract recovery was calculated.The percentage extract of P.santalinoides, M.sativa and G.latifolium were 13.5%, 11.1% and 7% respectively.They were stored in a freezer (≤4.0 0 C ) until needed.
= dryweight of extract recovered after extraction Initial dryweight of plant part × 100

Characterization of plant extract active component
Characterization of plant extract active component was carried out using GC-MS analysis by the method of 5 .

Preparation of sample
The leaves of the plant were air dried at room temperature for about 2 days.The dried leaves were ground dried and powdered samples were subjected to soxhlet extraction method.

Soxhlet Extraction method
Boiling flasks of 500ml were dried in oven at 105 -110 0 C for about 30 minutes.It was transferred into a dessicator and allowed to cool.Sample of 100g was poured into the Soxhlet thimble.The boiling flask was filled with about 300ml of ethanol and allowed to reflux for about 4 hours at a temperature of (60 0 C).The extract was poured into a volumetric flask and allowed to cool.Then, rotatory evaporator was used to separate the solvent (n-hexane) from the oil.

Extraction of phytochemicals
Extract of 1g was weighed and transferred in a test tube and 25ml of ethanol added.The test tube was allowed to react in a hotplate at 60 0 C for 90mins.After the reaction time, the reaction product contained in the test tube was transferred to a separatory funnel.The tube was washed successfully with 20ml of ethanol, 10ml of cold water, 10ml of hot water and 3ml of hexane, which were all transferred to the funnel.The extracts were combined and washed three times with 10ml of 10%v/v ethanol aqueous solution.The solution was dried with anhydrous sodium sulfate and the solvent was evaporated.The sample was solubilized in 1000ul of pyridine of which 200µl was transferred to a vial for analysis.

Quantification by GC-MS
The analysis of phytochemical was performed on a BUCK M910 Gas chromatography equipped with HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm in thickness of film).Spectroscopic detection by GC-MS involved an electron ionization system which utilized high energy electrons (70 eV).Pure helium gas (99.995%) was used as the carrier gas with flow rate of 1 mL/min.The initial temperature was set at 50 °C with increasing rate of 3 °C/min and holding time of about 10 min.Finally, the temperature was increased to 300 °C at 10 °C/min.One microliter of the prepared 1% of the extracts diluted with acetonitrile was injected in splitless mode.Relative quantity of the chemical compounds present in each of the extracts was expressed as percentage, based on peak area produced in the chromatogram.

Identification of chemical constituents
Bioactive compounds extracted from different extracts were identified based on GC retention time on HP-5MS column and matching of the spectra with computer software data of standards (Replib and Mainlab data of GC-MS systems) 6 .

Fourier Transform Infrared Spectrophotometer (FT-IR)
Buck scientific M530 USA FTIR was used for the analysis.This instrument was equipped with a detector of deuterated triglycine sulphate and beam splitter of potassium bromide.The software of the Gram A1 was used to obtain the spectra and to manipulate them.An approximately of 1.0g of samples, 0.5ml of nujol was added, they were mixed properly and placed on a the salt pellet..During measurement, FTIR spectra was obtained at frequency regions of 4,000 -600 cm-1 and co-added at 32 scans and at 4 cm-1 resolution.FTIR spectra were displayed as transmitter values.
Table 5 shows the result for P.santaiinoides sample, the peak value around 755.7627cm -1 was assigned to C-CI stretching vibration of chloro compound.The absorbance around 1161.138cm -1 was assigned to C-0 stretching vibration of ether compound.The adsorption band around 1690.529cm -1 was assigned to N-H stretching vibration of 1 0 amine compound whereas the spectra height around 1875.572cm -1 was assigned to C-0 stretching vibration cyclic ester compound.The absorption band around 2032.003cm -1 and 2109.049cm - were assigned to C00 stretching vibration of carboxylic acid compound respectively.The peak around 2258.558cm -1 was assigned to C-0 stretching vibration of carbonyl compound.The peak value around 2461.492cm -1 and 2593.730cm - corresponds to C-N stretching vibration of nitrile compound respectively.The weak band around 2791.497cm -1 was assigned to C-H stretching vibration of methylene compound.The strong band around 3020.363cm -1 , 3236.081cm -1 , 3361.486cm -1 , 3538.358cm -1 , 3690.035cm -1 and 3814.708cm - 1 corresponds to 0-H stretching vibration of 1 0 , 2 0 & 3 0 alcoholic compounds respectively.
From the table of results for M.sativa sample (Table 6), the peak value around 825.7247cm -1 was assigned to C-CI stretching vibration of chloro compound.The absorbance around 1143.633cm -1 was assigned to C-0 stretching vibration of ether compound.The peak around 1305.183cm -1 and 1412.511cm-1 were assigned to C=C stretching vibration of ethene compound respectively.The adsorption band around 1615.427cm -1 was assigned to to N-H stretching vibration of 1 0 amine compound whereas the spectra height around 1858.289cm -1 was assigned to C-0 stretching vibration cyclic ester compound.The absorption band around 2061.864cm -1 was assigned to C00 stretching vibration of carboxylic acid compound.The peak around2268.355cm - was assigned to C-0 stretching vibration of carbonyl compound.The peak value around 2449.902cm -1 corresponds to C-N stretching vibration of nitrile compound.The weak band around 2600.965cm -1 and 2811.642cm - were assigned to C-H stretching vibration of methylene compound respectively.The peak around 2977.171cm -1 was assigned to SCN stretching vibration of thiocyanate compound.The strong band around 3275.852cm -1 and 3395.574cm - corresponds to 0-H stretching vibration of 1 0 , 2 0 alcoholic compounds respectively.The rich chemical composition of catechin, flavon-3-ol, sparteine, resveratrol, 9,12-octadecadienoic acid, aragyrine, baptifoline, 6-octadececanoic, and kaempferol was revealed by the characterization of the active components of Medicago sativa, Gongronema latifolium, and Pterocarpus santalinoides (Table 1,2,3).Tea's primary functional ingredient, catechins, has various health advantages, including the reduction of sugar in diabetes.By enhancing insulin resistance, reducing oxidative stress, regulating mitochondrial function, reducing endoplasmic reticulum stress, providing anti-inflammatory effects, lowering blood sugar, and regulating intestinal function, catechins can alleviate hyperglycemia 14,15,16 .According to studies, catechins reduce fasting blood glucose levels in diabetic rats and inhibit carbohydrate-digesting enzymes including -amylase and -glucosidase 17 .By regulating gut microbiota, modifying inflammatory response, suppressing oxidative stress, and boosting immune function, flavon-3-ol reduces the severity of diabetes complications like retinopathy, nephropathy, neuropathy, vascular dysfunction, cardiomyopathy, coronary diseases, and renal failure.By preserving pancreatic beta cells, increasing insulin signaling, encouraging the pancreas to release insulin, blocking glucogenolysis, inhibiting gluconeogenesis, inhibiting digesting enzymes, and inhibiting carbohydrate metabolizing enzymes, flavon-3-ol lowers blood sugar levels 8 .In type 2 diabetics, L-arginine increased glucose levels, insulin-induced hepatic glucose synthesis, and insulin sensitivity 11 .The study discovered the existence of decanoic acid, a saturated fatty acid that regulates the activity of PPAR receptors, a subfamily of nuclear receptors that is essential for the metabolism of lipids and glucose.Treatments with decanoic acid and its triglyceride form enhance lipid profiles and glucose sensitivity in diabetic animals without causing weight gain 13 .Significantly less miRNA-375 is expressed when resveratrol is present.MiRNA-375 may help treat diabetes mellitus by preventing the body's beta cells from dying through apoptosis, which would maintain enough insulin output in the body 10 .Through PI3K/AKT signal pathways, resveratrol ameliorates uric acid-induced pancreatic beta-cell damage and increases insulin secretion 16 .Sparteine decreases the K+ permeability of the B-cell membrane to promote insulin release 9 .Sparteine caused an electrical activity similar to that of glucose, depolarized the B-cell membrane, and prompted the release of insulin.Similar to an insulin secretagogue, kaempferol encourages insulin secretion.In a study employing the insulin secretagogue glibenclamide as the control substance, it was discovered that kaempferol raised plasma insulin levels and lowered blood glucose in streptozotocin-induced diabetic rats 12 .Mitochondrial Ca 2+ is crucial for the release of insulin and the metabolism of glucose.The primary mechanism for Ca 2+ uptake in mitochondria is the mitochondrial calcium monoporter (MCU).
Perhaps the most effective tool for determining the kinds of chemical bonds/functional groups that are present in the phytochemicals is the Fourier Transform Infrared Spectrophotometer (FT-IR).As observed in the annotated spectrum, the wavelength of light absorbed is a key aspect of the chemical bond.It is possible to identify the chemical bonds in a substance by reading its infrared absorption spectra 19 .The current FT-IR results are in agreement with earlier studies done on other plants and confirmed the presence of alkanes, alkenes, amines, carboxylic acids, and alcohols in the three plants 20,21,22,23 .Numerous studies have shown that the FT-IR spectrum is a useful tool for identifying, categorizing, and distinguishing between groups of closely related plants and other species.

Conclusion
Alcohol, alkanes, and ester molecules were identified as functional groups of OH, C-H, and C0O in this plant specimen.The chemicals discovered during GC-MS analysis of these plants extract support the traditional practitioner's use of them in the treatment of diabetes mellitus.However, since the detected compounds were previously reported to have significant bio-activity, isolating of each individual component and testing it for biological activity will undoubtedly produce positive results.As a result, using this plant as a source of phytopharmaceutical value is advised.

Disclosure of conflict of interest
No conflict of interest to be disclosed.

1 .
Characterization of the plant active chemical components and determination of the functional groups.

Table 2
Chemical Constituents of Pterocarpus santalinoides leaf extract

Table 3
Chemical Constituents of Medicago sativa leaf extract

Table 4
Functional groups content of Gongronema latifolium leaf extract

Table 5
Functional groups content of Pterocarpus santalinoides leaf extract

Table 6
Functional groups content of Medicago sativa leaf extract