INTRODUCTION:

Today's inactive lifestyle and nutrient-deficient diets invites various and serious diseases and disorders. Diabetes, cardiovascular and hypertension are the most life-threatening disorders.¹ To overcome and minimize these, we need to have nutrient-rich food habits to follow. It can be fulfilled by consuming raw vegetables with dietary components. It will help to mitigate serious disorders. Charantin is nothing but an equal composition of stigmasterol and sitosterol glucosides.²

Type II Diabetes mellitus (DM2) is a growing disorder in the world day by day that is becoming impossible to control. Various researchers studied the effectiveness of charantin against type II diabetes.^3,4 Richter⁵, studied effectiveness against type II diabetes and Alzheimer's disease. Instead of going for allopathic treatment and to avoid side effects and additional complications while on medication, a natural source of diabetes control is essential. Bitter gourd works effectively to control oxidation of cells, cell mutation, antitumor, controls inflammatory processes, and anticarcinogenic.⁶ Potentially bitter gourds are very important in type II diabetes management.⁷ Bitter gourd contains Charantin, a compound which has the capability to control blood sugar. Alcoholic extract of bitter gourd consumed.⁸ Bitter gourd charantin is an essential triterpenoid, available in bitter gourd fruits which is used as an antidiabetic to control the blood sugar of a diabetic population.⁹ Charantin is also known as saponins, as alkaloids with potent hypoglycemic activity.¹⁰ Investigated various charantin rich extracts as antihyperglycemic activity.¹¹ Bitter gourd is a great source of antioxidants¹² like betle leaf.¹³ Some researchers studied bitter gourd charantin for both type 1 and type II diabetes clinical studies in mice¹⁴ Bitter gourd found a panacea against cancerous and inflammatory cells.¹⁵ To study functions, characterization of phytomolecules like charantin becomes emerging concept¹⁶

MATERIALS AND METHODS:

Plant Materials and Methods Fruit sample and its type:

Bitter gourd seeds 10 accessions were grown at the Mandsaur University, Mandsaur Madhya Pradesh, India. All to be tested accessions grown at standard agronomic practices for sample preparation.¹⁷ Well matured and uniform fruits in shape and size collected for further process of fresh fruit cutting(figure 1a), drying (figure 1b) and grinding (figure 1c). Fruit morphological characters like fruit shape, size, ripening, maturity are very much important in level of nutritional attributes¹⁸

Figure 1: Sample preparation images-a) Collected fresh fruits, b) Dried slices and c) Ground powder

Sample preparation:

Fresh bitter gourd fruits were collected as 2kg from each accession from the field. Fruits with over and below maturity, deformed, virus infected were removed to get proper maturity fruits. To avoid inert matter, all fruits were cleaned thoroughly with tap water and then distilled water. Cut fresh whole fruits with seeds into small slices. Drying of fruit slices is the most important step before charantin extraction to avoid interference of water soluble impurities. There are various methods used for drying like infrared, hot air oven, freeze drying, sun drying and hybrid methods as well.¹⁹ Fresh bitter gourd slices (chopped) were sun dried in a plastic tray for 6-7 hours under sunlight for 3 days to dry completely. 85-90 % water content was reduced in bitter gourd samples before drying.²⁰

Table 1: Extraction parameters and solvent

Solvents Parameters	Absolute methanol	Absolute ethanol	Ethanol + Methanol (1:1)
Solvent quantity in ml	200	200	200
Sample type	Dry powder	Dry powder	Dry powder
Sample quantity in grams	20	20	20
Time in hrs.	5	5	5
Temp in ℃	64.7	78.37	73.5
Instrument	Soxhlet	Soxhlet	Soxhlet

Chemical Materials and Method Soxhlet extraction:

As shown in table-1, 20 gram bitter gourd powder, 200 ml solvent quantity with different temperature used to extract the charantin in respective solvents for five hours. Different solvent combinations used to understand the reasonable solvent for maximum amount of charantin to be extracted. Used soxhlet (Kumar Biotech, India) as in Figure 1a. We have selected this method due to its cost effectiveness, robustness and availability. It is assembled with three different parts, condenser, extractor and flat bottom flask for extraction. All the extracts of ethanol, methanol and mixture of ethanol were collected in flask, filtered with whatman number1 paper.²⁰ All three extracts were evaporated in a hot air oven to get 20 ml concentrated extract for quantification. The extract was stored at controlled condition at 4-8℃ in the refrigerator till in use of analysis (figure 2b).

Figure 2: a) Soxhlet charantin extraction b) Filtered extract

HPLC Method Development and parameters:

The extract filtered through Whatman filter paper, again filtered with a syringe filter with pore size 0.45um and filled in HPLC vials. Vials loaded in HPLC machine for analysis and separation using modified parameters (Table 2). Adjusted column oven, temperature, pressure, before preheating, purging and analysis run started.

Table 2: HPLC conditions

Parameters	Specification
Stationary phase	C18 column (75 x 4.6 mm x 5 particle size)
Mobile phase A	80 % Methanol (HPLC grade)
Mobile phase B	20 % Acetonitrile (HPLC grade)
Detector	UV detector
Sample Run time	10minutes
Flow rate	0.7ml/min
Injection volume	10ul

RESULTS AND DISCUSSION:

Effect of fruit maturity, drying and moisture:

Collected well matured fruits of 14-15 days fruit maturity having better charantin extraction because of its biochemical expression. Immatured and over matured fruits with incomplete synthesis or degradation of charantin respectively. On sun drying observed moisture content from 11% to 14%.

Moisture % Calculation:

% Moisture (M) content = M1-M2

M1-M0

Where,

M0 is weight of empty glass plate

M1 is weight glass plate with sample before drying

M2 is weight of sample with plate after drying

Effect of boiling temperature and concentrated extract:

Soxhlet extraction in/is a continuous hot extraction process²² and works like reflux with extracts. Boiling temperature is the major factor affecting the extraction of optimum charantin in bitter gourd fruits. Charantin extraction occurs effectively in methanol because of medium boiling point (Compared to absolute ethanol and ethanol:methanol combination). It indicates that more charantin extracts from bitter gourd from/at temp 65-68℃ but above 68℃ and below 66℃ it extracts more impurities which interferes in getting chromatograms.²³

Bitter gourd charantin HPLC analysis:

HPLC parameters modified/adjusted and implemented for method development.^24,25,26 HPLC system (Prominence i series, Shimadzu Co., Japan) loaded with software and UV detectors. The stationary phase C18 silica column with specification 75 × 4.6mm x 5 particle size. Applied HPLC parameters, mobile phase and other conditions as shown in table 2. Sample aliquots were filtered through a 0.45µm poly filter prior to injection. Identification and quantification of charantin was carried out by comparing the retention times and the peak areas converted to ug/g dry weight (Table 3) by using the equation

Concentration of sample = Area of sample/Area of standard x concentration of standard.

Table 3: Charantin concentration in ug/g dry weight

Charantin conc. in ug/g
Ethanol	Methanol	Ethanol + Methanol
246.358	385.697	322.302
196.030	369.314	317.508
143.402	283.920	159.790
505.210	837.480	687.251
611.364	919.173	659.507
664.811	927.016	780.807
568.058	677.484	598.264
326.758	571.302	330.388
447.507	701.994	520.824
373.485	920.338	505.559

CONCLUSION:

This research study can conclude that methanol might be the best solvent for extraction of maximum and pure charantin from fresh bitter gourd fruits on drying at optimum temperature and pH of the solvent in soxhlet. Blanching also helped to avoid loss of nutrients at storage.

The present study conducted to get the most suitable solvent for the extraction of charantin which results in a maximum amount of charantin in a quantified way. Used food grade organic solvents for making charantin rich extract to utilize it for type II diabetes. This extraction solvent method and quantitative data help to compose and formulate charantin rich raw product with some additional antidiabetic herbs and spices.In the future it might become a great platform to formulate a raw products in the form of tablet, capsule or raw powder to use against type II diabetes. Only disadvantage is that it would be perishable so it would be consumed as soon as possible.

CONFLICT OF INTEREST:

Author declares that there is no conflict of interest.

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Received on 01.06.2024 Revised on 18.10.2024

Accepted on 19.12.2024 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):2957-2960.

DOI: 10.52711/0974-360X.2025.00423

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