Momordica charantia L. is a plant of the Cucurbitaceae’s family, found in African wild flora. It is widely spread throughout tropical African area and is taken for the wild form of the species. It is harvested in a natural state and used as a vegetable or medicinal plant (Schmelzer and Gurib-Fakim, 2008) (Cantwell et al., 1996).  

In Congo, this plant is used in traditional medicine as antidiabetic and for its many other medicinal virtues (antimalarial, deworming, laxative, etc...). The ethnobotanical survey conducted in Brazzaville (Congo) by Ampa and al showed that M. charantia L. is a widely used antidiabetic plant (Ahombo et al., 2012; Adjanohoun et al., 1988). Although Congolese traditional healers prescribe the plant to diabetics, no antidiabetic and phytochemical studies have been made on the species present in Congo Brazzaville. Studies performed on this plant in China and India have revealed the presence of flavonoids, tannins, alkaloids, cardiac glycosides, saponins and steroids. More molecules like momordicosides, momordenol, momordicin, cucurbitacin, charantine were identified and isolated and among them some tested antidiabetic compounds such as antidiabetic activities of  vicine, polypeptide P, charantine and many other triterpenoid (Singh et al., 2012; Majekodunmi et al., 1990; Sabira et al., 1997; Kimura et al., 2005; Sonal and Pratima, 2015; Haixia et al., 2004). Charantine, stigmasterol and β-sitosterol glucoside steroidal saponins with effective antidiabetic properties. Some studies have shown that M. charantia has insulin secretory properties (Kedar and  Chakrabarti,  1982).  Several  Works  in  Asia highlight that it is possible to extract charantine in seeds, in fruits, and in leaves of M. charantia L., by chromatographic and spectral methods such as high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), ultraviolet spectrometry, infrared spectrometryHaixia Z, Xiaozuo Z, Yawei W, Mancanq L, Zhide H, 2004).

This research focuses on evaluating the antidiabetic activity of the ethanolic extract of the M. charantia L. in Congo and to verify the presence of the charantine (antidiabetic molecule) by a method using gas chromatography coupled with mass spectrometry. The purpose of this paper is to confirm the antidiabetic activity of M. charantia species found in Congo Brazzaville.

Plant material

Aerial parts from M. charantia L. (Figure 1) were collected in MFILOU a southern district of Brazzaville (Congo). Botanical identification of the plant was made in the Research Institute of Exact and Natural Sciences (IRSEN). The voucher specimen deposited in the national herbarium is Mampouya C. Mounsambote JM # 201 August 10, 2017.

(i) for sample preparation a soxhlet extractor, a BUCHI rotary evaporator;

(ii) for sample analysis a Hewlett Packard 6890 gas chromatograph coupled with MS Engine Hewlett Packard 5973 quadrupole mass spectrometry;

(iii) for evaluation of the antidiabetic activity: a glucometer of brand one touch with strips.

Preparation of the ethanolic extract

Aerial parts of M. charantia L. were dried in a dry place away from the light for a week and then crushed. The powder was stored in a glass vial. 20 g of the powder obtained were put in a cellulose cartridge and then extracted for 3 h with ethanol in a Soxhlet extractor with a capacity of 250 ml. The resulting extract was dried using a rotary evaporator to obtain a dry ethanolic extract.

Biological methods

Induction of diabetes

Male rats of an average weight between 200  and 350 g were anaesthetised with diethyl ether and then made diabetic by intravenous injection of streptozotocin (Sigma Aldrich) freshly prepared in sodium chloride buffer solution (0.9 %), at 55 mg / kg. Rats’ Glycemia was monitored 72 h later. Rats with glycemia higher than 1.40 g/L were selected for the experiment (Andrade-Cetto et al., 2000).

Evaluation of the antidiabetic activity

The diabetic animals were fasted for 12 h and divided into three lots of five rats each. These different lots received respectively: distilled water (10 ml/kg),  Glibenclamide  (10 mg/kg)  and  ethanolic  extract (400 mg/kg). Glycemia was taken every 4 h. A drop of blood collected by making a slight incision of the distal end of the tail of the rat was deposited on the reactive range of the strip. The reading is done on the glucometer which displayed the results in mmol/ml, then they were converted into g/L. 

Statistical analysis

The results of the blood glucose are presented in the average form ± average standard error. Variance analysis (ANOVA) and the Student's test were used for the statistical study. The number of experiments in each group was n = 5. At the P <0.05 threshold, the values ​​of blood glucose variation were considered significant.

Chemical methods

Methods of analysis

Treatment of the ethanolic extract: 2 mg of the dry ethanolic extract were added to 500 μl of the bypass reagent (pyridine / MSTFA) and then placed in an oil bath at 80°C for 2 h. The reagent was then evaporated to dryness using a rotary evaporator, the residue is taken up with 500 μl of hexane. The solution obtained is then ready for analysis in gas chromatography coupled with mass spectrometry (GC-MS) in split mode.  10 μl of the sample were injected. The duration of the analysis was 120 min, the acquisition began at 25 min.

Analysis of the extract in GC-MS: The operating conditions and the parameters of the chromatograph coupled with the mass spectrometer for the analysis of the ethanolic extract after derivatization are shown in Tables 1 and 2. The mass spectra are recorded in electronic impact mode with an ionization energy of 70 eV in "full-scan" mode. Chemical identification is carried out by comparing  the  mass spectrum obtained with that of reference. The software used x callibur.

Effeciency of extraction

The extraction of 20 g of M. charantia L aerial parts with ethanol gave a crude weighing 2.34 g, which was a 2.34 g, and yield of 11.7%.

Effect of streptozotocin on normal rats

After injection of streptozotocin, polyuria and elevation of fasting rat glucose were observed with values ​​between 1.40 and 2.53 g/L. These parameters justify that the rats were indeed made diabetic.

Evaluation of the antidiabetic effect of the ethanolic extract of M. charantia

Table 3 presents the evolution of the mean blood glucose level with percentages of glycemic reduction in diabetic rats that have been orally treated  with  ethanol  extract,  Glibenclamide  and distilled water at the doses indicated above. As shown in the table, only the rats that received glibenclamide and plant extract showed significant reductions in blood glucose levels in the first hour, respectively 22.73 and 29.67% versus 8.99% for those that received distilled water. The latter showed no significant reduction in blood glucose until the fourth hour, while the other two lots of rats showed very significant reductions in blood glucose levels until the fourth hour (p <0.05). At the fourth hour, the percentage reduction in glycemia of rats treated with glibenclamide continued to increase (58.44% at the third hour for 59.10% at the fourth hour), while that of the rats treated with the extract plant starts to decline (51.62 to 47.56% at the fourth hour).

GC-MS analysis of M. charantia L. ethanolic extract and Identification of sterol glucoside: charantin

The exploitation of the chromatogram with the database available in the laboratory enabled us to identify at different retention times several compounds and particularly β-sitosterol glucoside (charantin) with a retention time of 73.84 min (Figure  3).  Mass  spectrum  related  to  the  peak eluted at 73.84 is shown in Figure 2. On this mass spectrum, the following can be observed:

(i) The base peak is at m / z is 204,09 ;

(ii) Peaks of 395,34 and 451,10 are fragments of sterol and glucoside respectively

(iii) Ions at m/z : 147; 204; 217; 305 and 361 are characteristic of the carbohydrate moiety;

(iv) m/z : 129 and 255 are peaks characteristic of the sterol part; 

(v) The molecular peak is that of which m / z = 861.43.  

All of these spectral data are oriented towards a steroid compound whose basic skeleton is a stigmasterol; m/z = 395.34 (Figure 3) stuck to a sugar: glucose; m/z = 451.10 (Figure 4). The steryl glycoside TMSi ether allowed it to be analysed by GC/MS on a short packed column or by a capillary column operated at high temperature. The mass spectrum of steryl glycoside TMSi ether shows, molecular peak is not perceivable and it is dominated by ions arising from hexose TMSi ether moiety (m/z 361, 217, 204, 191 and 147). The base peak for a hexose pyranoside  moiety is at m/z 204 but for a furanoside the prominent ion is m/z 217. The presence  of stigmasterol glucoside (charantin) by GC-MS analysis of the ethanolic extract of the leafy stems of M. charantia was confirmed on the basis of the comparison of the mass spectrum obtained with the work of Phillips KM. In addition, Figure 3 shows the characteristic mass spectrum of sterol glucoside peaks. Characteristic peaks of the steroid fragment were also observed in this spectrum (Phillips et al., 2005; Goad and Akihisa, 1997).

The data from the chromatogram and the mass spectrum obtained made it possible to emphasize the presence of charantine in the ethanolic extract of M. charantia L. with structure as shown in Figure 5.

Previous    work    has   shown   that   the   isolation   and identification of charantine is performed using several techniques such as: HPTLC, LC-MS or HPLC-DAD (Sonal and Pratima, 2015; Thomas et al., 2012). This work identified this compound in the various organs (leaf, fruit and seed) of M. charantia L., a species harvested in China and India using techniques such as HPTLC, LC-MS and HPLC-DAD (Ahamad et al., 2014). However, to date, analytical and phytochemical studies to identify antidiabetic compounds in the species present in Central Africa have not yet been carried out. 

The bibliographic data showed that charantine has anti-diabetic properties (Sonal and Pratima, 2015). In the Republic of Congo (RC), Chad and Benin, the plant is used in traditional medicine to treat diabetes. The presence of this molecule could justify its use in traditional medicine against diabetes (Sakine et al., 2014; Ahombo et al., 2012; Lalèyè et  al., 2015). The charantine has been isolated in the seeds, fruits and leaves of M. charantia. The molecule has been tested to treat diabetes. The work by Sonal and Pratima (2015) showed that at the dose of 50 mg / kg, the analyses give a 42% reduction on glycemia percentage after 4 h, this percentage is reduced at the 5th hour by 28% (Lalèyè et al., 2015; Pitiphanpong et al., 2007). The ethanolic extract of M. charantia aerial parts administered at a dose of 400 mg / kg in male rats gave significant antidiabetic activity. This extract caused a drop in blood sugar until the 3rd hour but we observe at the 4th hour a slight increase in blood sugar. Previous work by Nagy (2012) has shown that the aqueous extract and even the ethanolic extract of M. charantia L. reduces blood glucose levels in type 2 diabetic rats. Other studies have suggested the existence of charantine having an effect on blood glucose (Nagy et al., 2012). 

The results obtained make it possible to point out that the ethanolic extract of M. charantia decreases blood glucose at a dose of 400 mg / kg, which shows that the plant has antidiabetic effects. These effects may be due to the presence of certain chemical components such as flavonoids, alkaloids, saponins (Day et al., 1990; Coskun et al., 2005; Han et al., 2008; Neha et al., 2016). The work by Jesada et  al.  (2007)  on  the  M.  charantia  was able to isolate and highlight the charantine, besides the results by Wang has shown that this molecule has antidiabetic virtues more particularly insulinosécrétrices properties (Pitiphanpong et al., 2007; Wang et al., 2014). The presence of charantine in our species could justify the reduction of glycemia in diabetic rats treated with extracts of M. charantia and the use of this plant as an antidiabetic in Congolese traditional medicine.  

The aerial parts of M. charantia L. are in common use in traditional Congolese medicine for their multiple virtues. In addition to the current use of M. charantia L. decoction, the ethanol extract also has considerable antidiabetic activity. The present study based on the demonstration of the antidiabetic properties of the ethanolic extract of M. charantia L. and also on the identification of a recognized antidiabetic molecule: the charantine. The charantine identification was made by gas chromatography coupled to the mass spectrometry of the extract of the aerial parts of M. charantia L. var abreviata harvested in Brazzaville (Congo), after treatment with MSTFA. The separation and identification of the compounds was carried out on a column consisting of 5% phenyl-95% methylpolysiloxane. All mass spectra were recorded in electronic impact with an ionization energy of 70 eV. The ethanol extract, tested by injection of streptozotocin on albino Wistar rats made diabetic, induced a significant reduction in blood glucose (51.62%) at the 3rd hour. The significant results of the antidiabetic tests and the demonstration of the charantine justify the use of this plant in traditional medicine by the traditional healers in Congo Brazzaville.

The authors have not declared any conflict of interests.

This study is financially supported by the SCAC of the French Embassy and carried out in the UC2V and LBP laboratories of the University Marien Ngouabi - Congo, and LETIAM (Laboratoire d’étude des techniques et instruments d’analyse moléculaire) of the IUT (Institut universitaire de technologie) of Orsay France. We thank Professor Jean Maurille Ouamba, the initiator of this research project, and Professor Alain Tchapla for his coordination and support during our experiments at LETIAM (IUT d'Orsay) and Professor Jean Marie Mounsambote for the identification of the plant.