INTRODUCTION:

The versatility of Mucuna species is apparent in a range of pharmacological activities. In-vitro testing revealed the presence of antibacterial properties¹. An assessment of anticancer potency revealed that it has remarkable effects on lung cancer cells². The analgesic, anti-inflammatory, antispasmodic, aphrodisiac, febrifuge, hypoglycemic, immunomodulatory, antilithiatic, blood purifying, carminative, hypotensive, and uterine stimulating activities of Mucuna species seeds are extensively documented³. Furthermore, antivenom and anthelminthic properties have been reported as well⁴.

It has been reported that the seeds of Mucuna cochinchinensis (M. cochinchinensis) exhibit strong antioxidant activity⁵. As M. cochinchinenisis lacks a specialised characteristic of stings on the pods, its distinctive morphological characteristics caught our attention.

The nutritional value of M. cochinchinensisis is better than that of other legumes and comparable to that of soybeans⁶. Many legumes have multiple uses, including as food, fodder, and pharmaceuticals⁷. For their prospective use in the pharmaceutical and nutraceutical industries, the variations in the genus Mucuna require further focused attention and analysis by the researchers. Diabetes mellitus, a metabolic disorder characterized by hyperglycemia and improper lipid, carbohydrate, and protein metabolism, has one of the highest rate of prevalence in the world⁸. Many reports have been made on anti-diabetic properties of phytochemical extracts from certain plants of biological importance^9-16. There are numerous pharmacological actions, responsible bioactive components, and associated mechanisms in M. cochinchinensis that need to be determined.

As a consequence, it was decided to evaluate the plant in this study in order to determine its pharmacological potential to attenuate the signs of diabetes mellitus.

MATERIALS AND METHODS:

Plant Collection and authentication:

The matured seeds of M. cochinchinensis were collected from Western Ghats, Mundanthurai, Tamilnadu during the month of February after winter season. The flowering occurred in the month of December and the pods matured during the month of February. During collection, care was taken to select healthy, normal and even size seeds. The collected seeds were authenticated by renowned botanist.

Preparation of plant extract:

The seeds were dried in shade and powdered coarsely using a mechanical grinder. About 250 g of coarse seed powder was extracted by cold maceration using shaker for 72 h in 1L of ethyl acetate. The ethyl acetate extract of M. cochinchinensis (EMC) was concentrated in rotary vacuum evaporator and preserved in a tightly closed container¹⁷.

Preliminary phytochemical analysis:

Preliminary phytochemical analysis was carried out for understanding the different chemical components present EMC¹⁸.

HPLC/ESI-MS/MS:

Liquid chromatography/ electron spray ionization/ mass spectroscopy (LC/ESI/MS) was used to separate and qualitatively analyse phyto components present in EMC using a Bruker UHPLC 3000 chromatograph coupled to a quadrupole-ToF mass selective detector (microToF QII, Bruker, Germany). The extract was chromatographically separated using an RP-C18 column from Acclaim®, Sunnyvale, California, USA (100mm × 3.9mm, internal diameter: 5 µm).The following gradient profile was employed in gradient elution using mobile phase A, represented by acetonitrile, and mobile phase B, represented by MilliQ water acidified with acetic acid (2%): Initially 95% B to 80% B over 5min; to 70% B over 5min; to 65% B over 5min; to 40% B over 5min; to 0% B over 5min; to 95% B over 2.5min until the run is completed¹⁹. The flow rate was set to 200µL/min, and UV absorbance at 335 nm was detected. In negative ion mode, a mass spectrum was generated. The HPLC-MS/MS technique was employed to identify the ligand molecules, and the PubChem small molecular database was used to retrieve their structures²⁰.

Anti-diabetic screening:

Wistar rats with diabetes were used to test the anti-diabetic effects of EMC in-vivo. Alloxan was used to induce diabetes in the study rats^21-23. On days 1, 7, and 14, the blood glucose level was estimated. Additionally, plasma insulin levels were assessed at end of the investigation.

Animals:

For the investigation, male Wistar rats weighing 150–200g were utilised. The animals were maintained in an animal house and were fed with standard rat feed (provided by Kamadenu Agencies in Bangalore, India) with water ad libitum. Standard conditions for animal maintenance were a 12 h light/dark cycle, a temperature of 25°C±2°C and a relative humidity range of 45–60%. Prior to the start of the studies, all the animals were acclimatized to laboratory conditions for a week. All experimental protocols were approved by the IAEC and laboratory animals were maintained with utmost care as stated in the CCSEA (earlier CPCSEA) regulations.

Experimental design:

Healthy Male Wistar rats were selected for inducing diabetes. The animals were given a single intra-peritoneal injection of a newly prepared solution of alloxan monohydrate at a dose of 140mg/kg body weight to induce diabetes. After 48 h of alloxan treatment, a fasting blood sample (post-12 h feed) was obtained from the tail vein by intravenous puncture. The sample was subjected to blood glucose analysis. Animals were classified as diabetic whose fasting blood glucose levels ranged between 210 and 220mg/dl. These animals also showed polyuria, polyphagia, and polydipsia, which are established symptoms and signs of diabetes mellitus. Induction of diabetes was unsuccessful in few animals and the rats which showed fasting blood glucose less than 200mg/dL was excluded. The animals selected were divided into 5 groups with 6 animals in each group and were subjected to the following treatment as mentioned in Table 1.

Table 1: Grouping of animals and treatment plan

Group I (normal control)	Vehicle treatment (1% Tween 80, 0.5ml/100gm b.w.p.o.)
Group II (diabetic control)	Alloxan à Vehicle treatment
Group III	Alloxan àEMC (200mg/kg b.w. p.o.)
Group IV	Alloxan àEMC (400mg/kg b.w. p.o.)
Group V	AlloxanàGlibenclamide (5 mg/kg, b.w. p.o.).

Glibenclamide was used as a standard drug to compare the hypoglycaemic effect of EMC. The animals were treated with standard and test sample for 14 days period.

Estimation of blood glucose and plasma insulin levels:

Blood samples were drawn from the tail tip of fasted rats and blood glucose levels were assessed using glucometer blood glucose test strips. On days 1, 7, and 14 of the study, body weight was measured and fasting blood glucose levels were estimated. On day 14, blood sample was collected in heparinized tubes from overnight fasted rats though retro-orbital plexus under mild ether anesthesia for estimation of insulin. The insulin concentration was determined by insulin assay kit.

Statistical Analysis:

The treated groups were compared with the toxicant control groups, all results were expressed as mean±SEM of 6 animals in each groups. The results were analyzed statistically using one way ANOVA followed by Dunnett’s test, P<0.01 was considered as significant.

RESULTS AND DISCUSSION:

Preliminary phytochemical analysis:

A preliminary phytochemical analysis of EMC found the presence of xanthoproteins, phenols, tannins, alkaloids, flavonoids, and anthraquinones. M. cochinchinensisis is said to be high in phenolic and flavonoid contents⁵.

Hplc/esi-ms/ms analysis:

Recent studies suggest that a potent approach for the quick identification of the constituents in medicinal plants and their preparations is the combination of HPLC and coupled techniques, such as Electron Spray Ionization (ESI) or Diode Array Detector (DAD) and mass spectra (MS)²⁴. In this study, acetonitrile and aqueous acetic acid were utilized as the appropriate eluting solvents for the analysis of flavonoids in M. cochinchinensis ethyl acetate extract (EMC) for the qualitative analysis. Fig.1 demonstrates a chromatogram of the phytoconstituents.

All the MS/MS experiments in this study were performed in the negative ion mode. The compounds were confirmed by small molecular data bases and further fragmentation pattern was analyzed. The presence of 6-C-Pentosyl-8-C-hexosyl Apigenin was confirmed by comparing the mass spectrometric indices (Table.2) of the same compounds isolated from other plant sources²⁵. The fragmentation patterns obtained for kaempferol-3-O-rutinoside were compared with those reported in previous study and the patterns were matching. Intense peak at 285 and 284 confirmed the kaempferol-3-O-rutinoside (Table.2). These intense peaks were the resultant in formation of radical aglycone²⁶.

Antidiabetic activity:

Cut-off dose 2000 mg/Kg Body Weight for M.cochinchinensis ethyl acetate extract was concluded as LD₅₀ and one-tenth and one-fifth dose of LD₅₀ was considered for Antidiabetic testing²⁷. Numerous researchers have developed common techniques to induce insulin-dependent diabetes mellitus (IDDM), such as the use of alloxan and streptozotocin. Alloxan and glucose share structural and molecular similarities, which facilitates alloxan to enter beta cells via the glucose transporter 2 (GLUT2)²⁸. Alloxan in the circulation undergoes rapid uptake up by pancreatic beta cells. Alloxan undergoes reduction in presence of reducing agents inside the pancreatic beta cells and further gets re-oxidized back to alloxan. This cyclical process results in the production of superoxide radicals and reactive oxygen species (ROS). DNA fragmentation in beta cells and oxidative damage caused by the afore mentioned radicals’ results in their destruction²¹. Similar to alloxan, streptozotocin, a glucose analogue, also enters the pancreatic beta cell through GLUT2 and accumulates inside the cell. In contrast, DNA alkylation, cellular necrosis, and the induction of IDDM occur in streptozotocin-induced diabetes²⁹.

Calcium concentrations or membrane polarity in neurons are unaffected by alloxan. As a result, primary nociceptive neurons are not depolarized. Because streptozotocin directly affects nociceptive neurons, likely through regulating TRPV1 channel activation, alloxan may therefore be a superior option for animal investigations involving painful diabetic neuropathy³⁰. These facts led to the use of alloxan in this study to induce diabetes in experimental rats. As indicated in Table 3, EMC administered at 2 different doses was found to effectively normalize plasma insulin concentration and blood glucose levels.

HPLC/MS study confirmed Kaempferol-3-O-rutinoside compound's presence in EMC. Kaempferol-3-O-rutinoside, which was derived from a variety of plant sources, has been shown to have anti-diabetic properties as per Kashyap et al. in 2023 and numerous other researchers in their studies^31-33. Increased blood glucose levels, elevated plasma free fatty acid levels, dyslipidemia, and elevated levels of alkaline phosphatase and transaminases are all caused by insulin deficiency, instigating metabolic changes and disorders³⁴. Numerous studies have examined and reported that many Mucuna species have crude fiber contents that range from 6% to 26%^4,35,36. According to these studies, Mucuna plants are a rich source of dietary fiber and can therefore lower plasma glucose levels and the subsequent complications that follow in diabetic patients. Furthermore, soluble dietary fiber-rich food ingredients have a high viscosity and function efficiently than insoluble fiber to delay intestinal glucose absorption^{37, 38}. It is significant to note that the medicinal plant's inorganic components, which primarily consist of mineral elements, contribute to the enhancement of the plant's medicinal potential, including its hypoglycemic activity. M. pruriens is found to contain a number of important macro- and microminerals, including Na, K, Ca, Zn, Mg, P, Fe, Cu, and Mn⁴. These components could be associated to the glucose tolerance factor or the mechanism of insulin release, as described in various laboratory animals and in humans³⁹. In streptozotocin-induced diabetic rats, an ethanol extract of M. pruriens at a dose level of 200 mg/kg/day was effective in regulating blood sugar levels^{40, 41}. This study, hereby conclusively states that EMC lowers blood glucose levels in alloxan-induced diabetic Wistar rats.

CONCLUSION:

To conclude, the ethyl acetate extract of M.cochinchinensis has been found to have anti-diabetic effect, which could be attributed to the presence of phytochemicals such as Kaempferol-O-rutinoside and 6C-pentosyl-8C-apigenin. Additional work is required for better understanding in defining the mechanism of action of hypoglycaemia.

CONFLICTS OF INTEREST:

The authors have no conflict of interest.

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List of symbols and Abbreviations included in this study

EMC	The ethyl acetate extract of M. cochinchinensis
LC/ESI/MS	Liquid Chromatography/ Electron spray Ionization/ Mass Spectroscopy
HPLC–MS/MS	high-Performance liquid chromatography (HPLC)/ mass spectrometry
ad libitum	as much or as often as necessary or desired
IAEC	Institutional Animal Ethics Committee
CCSEA	Committee for Control and Supervision of Experiments on Animals
ANOVA	Analysis of Variance
SEM	Standard Error of the Mean
ESI	Electron Spray Ionization
DAD	Diode Array Detector
LD₅₀	Median lethal dose
IDDM	insulin-dependent diabetes mellitus
GLUT2	glucose transporter 2
ROS	reactive oxygen species
DNA	Deoxyribonucleic Acid
TRPV1	Transient receptor potential vanilloid 1
Na, K, Ca, Zn, Mg, P, Fe, Cu, and Mn	Sodium, potassium, calcium, zinc, magnesium, phosphorus, iron, copper, and manganese

Received on 22.03.2023 Modified on 03.08.2023

Research J. Pharm. and Tech 2024; 17(3):1185-1189.

DOI: 10.52711/0974-360X.2024.00184

Treatment	Blood glucose level in mg/dl			% Inhibition 14^th day	PI µU/ml
Treatment	1^st day	7^th day	14^th day	% Inhibition 14^th day	PI µU/ml
I - Normal Control	92.3 ± 1.6	92.4 ± 3.4	91.8 ± 4.8	-	18.1 ± 0.60
II - Diabetic control	246.00 ± 1.60^#	268.86 ± 4.63^#	273.66 ± 4.84^#	-	4.32 ± 0.20^#
III- EMC 200 mg/kg	255.33 ± 1.76	185.66 ± 4.48*	108.66 ± 2.02*	57.44	11.06 ± 0.26*
IV- EMC 400 mg/kg	258.86 ± 2.07	175.00 ±3.05*	102.10 ± 1.94*	60.55	13.6 ± 0.23*
V- Standard 5mg/kg	254.96 ± 3.04	172.30 ± 3.10*	95.36 ± 2.12*	62.59	14.2 ± 0.32*