ANTIMALARIAL ACTIVITY AND TOXICITY EVALUATION OF THE ALKALOID-RICH FRACTION OF MOMORDICA CHARANTIA FRUITS

ANTIMALARIAL ACTIVITY AND TOXICITY EVALUATION OF THE ALKALOID-RICH FRACTION OF MOMORDICA CHARANTIA FRUITS

Syamsudin Abdillah ^{* 1}, Yunahara Farida ², Kartiningsih ³, Ni Made Dwi Sandhiutami ¹ and Kusdiantoro Mohamad ⁴

Department of Pharmacology ¹, Department of Chemistry ², Department of Technology Pharmacy ³, Faculty of Pharmacy, Pancasila University, Jl. Srengseng Sawah, Jagakarsa, South Jakarta 12640, Indonesia.

Department of Embriology ⁴, Faculty of Veterinary Medicine, Bogor Agricultural University, Bogor, Indonesia.

ABSTRACT: Introduction: We investigated to an in-vitro and in-vivo evaluation of antimalarial and toxicity of the alkaloid-rich fraction of Momordica charantia fruits. Materials and Methods: In-vitro and in-vivo antimalarial activity tests of Plasmodium falciparum strain 3D7 culture were performed on Plasmodium berghei-infected mice using suppressive and prophylactic methods, and evaluation of fraction toxicity was conducted on zebrafish (Danio rerio). Results and Discussion: The study found that IC₅₀ value of the in-vitro test was 0.17 ± 0.12 µg/mL and anti-malaria test using a suppressive and prophylactic method at a dosage of 100 mg/kg/day was found to inhibit parasites by 74 and 73%, respectively. The evaluation of toxicity by the use of zebrafish revealed abnormalities in the formation of the heart (51.28%) and yolk sac (51.28%). Conclusion: Alkaloid rich-fractions have powerful in-vitro and in-vivo antimalarial activity, but they have teratogenic effects that can inhibit the formation of the heart and yolk sac.

Anti-malarial activity, Toxicity, Alkaloid rich- fractions, Bitter melon (Momordica charantia)

INTRODUCTION: Malaria is caused by parasites of the Plasmodium family and is transmitted by female Anopheles mosquitoes. There are four different species of human malaria (Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale), of which P. falciparum and P. vivax are the most prevalent and P. falciparum is the most dangerous.

Plasmodium knowlesi is a zoonotic plasmodium that is also known to infect humans. Between 2000 and 2015, the incidence of malaria among populations at risk fell by 37% globally; during the same period, malaria mortality rates among populations at risk decreased by 60%. It is estimated that 6.2 million deaths from malaria have been prevented globally since 2001. ¹

In 2011, the number of malaria cases in Indonesia was 256,592 people from 1,322,451 cases of suspected malaria blood preparations being examined, with annual parasite incidence (API) of 1.75 per thousand populations. This means that for every 1,000 persons in endemic areas, there are 2 people who have been infected with malaria.

The greatest burden of malaria is in the provinces of eastern Indonesia, where malaria has become endemic ². Currently, Indonesia Ministry of Health announced that malaria pre-elimination stage should be reached by 2020 and be free of malaria transmission by 2030 to achieve the goal of an Asia-Pacific free of malaria by 2030. ³

Natural products and derivatives of the natural product have been a good source of new biologically active compounds for centuries. The first effective treatment for malaria was the natural product quinine, an alkaloid isolated from the bark of the South American Cinchona tree in 1817. ⁴ Alkaloids are one of the major classes of natural products that exhibit antimalarial activity.

Indeed, quinine, the first antimalarial drug, belongs to this class. Over 100 alkaloids from higher plants were reported to demonstrate significant antimalarial activity in studies published between 1990 and 2000; some of which are more potent than chloroquine ⁵. The research was conducted to determine alkaloid compounds in bitter melon (Momordica charantia). Previous studies have found that extracts of bitter melon have the most powerful properties among any other plant extracts, which have been used as antimalarials in Sei. Kepayang area, Asahan, North Sumatra ⁶.

The objective of the Study: The objective of the study was to characterize the influence of alkaloid rich-fractions of bitter melon (Momordica charantia) of in-vitro and in-vivo evaluation of antimalarial and toxicity.

MATERIALS AND METHODS:

Preparation of Alkaloid-rich Fraction: Three hundred grams of dried Simplicia was alkalinized in 150 ml of 25% NH₄OH, then extracted using ml of methanol for 2 days, and then stored under room temperature. The liquid extract was filtered; the filtrate was thickened using a rotary evaporator. The thick extract was added with 15 ml of H₂SO₄ in aquadest and then filtered. The filtrate was extracted using petroleum ether. The liquid phase was added with 25% NH₄OH to obtain pH 9-10 and then extracted using chloroform. The chloroform was partitioned using aquadest up to normal pH (pH 7); the chloroform phase was taken and to obtain the alkaloid fraction.

Determination of Total Alkaloid:  Two grams of extracts were carefully weighed, then extracted using 20 ml of methanol and 2 ml of ammonia, heated above a water bath for 30 min, and then filtered. The extraction process was replicated until the positive reaction against the alkaloid was stopped (negative alkaloid). The extract was added with 10 ml of 1 N hydrochloric acid; the extract was evaporated to a volume of 5 ml, and then filtered in a separate funnel.

The filtrate was alkalinized using sodium hydroxide up to pH 10 and then filtered several times using chloroform until the alkaloid was perfectly extracted. The chloroform extract was evaporated at a temperature of 50 °C and then dried at a temperature of 100 °C to obtain a normal weight. The total alkaloid was weighed ⁷.

In-vitro Antiplasmodial Assay: P. falciparum 3D7, obtained from Tropical Disease Center, University of Airlangga, Surabaya was maintained in our laboratory with 5% hematocrit (human-type O-positive red blood cells) in complete RPMI 1640 medium (RPMI 1640 medium supplemented with 25 mM HEPES, 370 μM hypoxanthine, 40 μg/mL 1 gentamycin, 0.25 μgml^-1 Fungizone and 0.5% [w/v] AlbuMax II) in 60 mm petri dish by modified candle jar method ⁸. The culture was routinely monitored through geimsa staining of the thin smears. Standard drugs (artemisinin and chloroquine) and the fraction-rich alkaloid bitter melon (M. charantia) at different concentrations of 1, 10, 100 µg/mL were prepared in DMSO and then diluted to achieve the required concentrations.

The synchronized culture with parasitemia of 1.5 and 3% hematocrit was incubated in 96-well microtitre plate, predisposed to multiple concentrations of compounds/extracts for 48 h at 37 °C in a candle jar. Blood smears from each well were fixed in methanol, stained with giemsa’s stain and the numbers of infected RBCs per 5000 cells were counted.

The antimalarial activity of the test fraction was expressed as 50% inhibitory concentration (IC₅₀) determined from the dose-response curve by non-linear regression analysis (curve-fit) using Graph Pad Prism (version 4) software ⁹.

 In-vivo Antiplasmodial Assay:

Suppression Test Method: Male Albino mice (6 to 8 weeks old, weighing 20 ± 2 g) were used in the experiment. The mice were housed in standard macrolon type II cages in air-conditioned rooms at 22 °C, 50 to 70% relative humidity, fed with the standard diet and received water ad libitum. Plasmodium berghei strain ANKA was maintained by serial passage of the infected blood through the intraperitoneal injection (ip). The test protocol was based on the 4-day suppressive test ¹⁰.

Repository Test: The prophylactic activity of the extract was tested using the residual infection procedure. Adult mice of both sexes were weighed and randomized into five groups of six mice each. Group I mice was given 1 ml of normal saline. Groups II, III and IV were given 1, 10 and 100 mg fraction/kg of body weight orally, respectively while Group V mice received 5 mg of chloroquine /kg of body weight orally daily for 5 days. On the fifth day, all the mice were inoculated with a standard inoculum of 0.1 x 107 P. berghei berghei NK 65 infected erythrocytes. A thick film of blood smears was then made from each mouse 72 h after treatment and examined microscopically for parasitemia level ¹¹.

Embryo-toxicity and Teratogenicity Assay: The assay established by Dulay et al., (2012) was followed in this study. Embryos were exposed to the different concentrations (100, 200, 300, 400, 500, 600, 700, 800 and 900 ppm) of each fraction, and embryo water served as the control in the 12-well ELISA plate. Four embryos were assayed per treatment, and each treatment was replicated three times. The plates were incubated at 26 ± 1 °C ¹².

Toxic and teratogenic effects were examined using a compound microscope every after 12 h after exposure to treatment. Mortality, hatchability, heartbeat rate, and malformations were recorded. Death was defined as coagulated embryos and an absence of visual heartbeat. Morphological endpoint evaluation of zebrafish was based on the established parameters: lethal (coagulation, tail not detached, no somites, and no heart-beat), teratogenic (malformation of the head, tail, and heart, scoliosis, deformity of yolk, and growth retardation), and normal ^{12, 13}. The validity of the test was determined. Data were analyzed using analysis of variance (ANOVA) and compared using the Duncan’s Multiple Range Test (DMRT) at 5% level of significance. The Sirichai Statistics 6.07 program was used for analysis.

RESULTS AND DISCUSSION: To administer better drugs against resistant strains of P. falciparum (and recently P. vivax), there is a need to search for new molecules from new sources ¹⁵. Medicinal plants have in the past been the source of some of the most successful antimalarial agents, such as the quinolines and artemisinin derivatives, and can still serve as a source for drug discovery ¹⁶. Previous research, which was conducted in SeiKepayang area, Asahan, North Sumatera, using ethnopharmacological approach, found 16 species of plants, which had been used as antimalaria ⁶. In-vitro antiplasmodial activity test found an IC₅₀ value of 0.017 µg/mL, whereas the in-vivo test found an ED₅₀ value of 19.61 mg/kg BWT. Extract of bitter melon was found to have more powerful activity than that of any other plant ¹⁷. In-vitro antiplasmodial activities of some extracts and fractions of alkaloid are presented in Table 1.

TABLE 1: RESULTS OF IN-VITRO ANTIPLASMODIAL ACTIVITY TEST OF SOME EXTRACTS AND FRACTIONS OF BITTER MELON

The alkaloid concentration test showed the best results for the alkaloid fraction (10.31%), followed by the thick extract of ethyl acetate (9.51%), the thick extract of n-hexane (6.51%), and thick 96% ethanolic extract (4.44%). In-vitro antiplasmodial activity test using P. falciparum strain 3D7 showed the most powerful alkaloid fraction compared to any other extracts and comparable to chloroquine.

In-vitro antiplasmodial activity assay on the culture of P. falciparum strain 3D7 showed that the alkaloid fraction of bitter melon (M. charantia) had an IC₅₀ value of 0.17 ± 0.12 µg/mL, a value that was comparable to that of positive control, namely quinine and artemisinin, which had IC₅₀ values of 0.38 ± 0.15 µg/mL and 0.15 ± 0.13 µg/mL, respectively. A study conducted by Yousif (2014) showed a powerful antiplasmodial activity of the chloroform fraction of bitter melon (M. charantia) with an IC₅₀ value of 1.83 ± 0.029 µg/ml in the culture of P. falciparum strain MRC-2 ¹⁸. Chloroform is a semi-polar solvent that can attract alkaloid compounds. The alkaloid extracts obtained from medicinal plant species have a multiplicity of host-mediated biological activities, including anti-malarial, antimicrobial, antihyperglycemic, anti-inflammatory and pharmacological effects ¹⁹. Some alkaloids have antiplasmodial activities, which have been successfully isolated, including alkaloids from Cyclea barbata, C. atjehensis, Stephania pierrei, S. erecta, Pachygone dasycarpa, Curarea candicans, Albertisia papuana (Menispermaceae), Hernandia peltata (Hernandiaceae), & Berberisval diviana (Berberidaceae) that exhibited a wide range of biological potencies in antiplasmodial assays, and the majority exhibited some degree of cytotoxicity against human KB cells. More than half of the tested compounds showed selective antiplasmodial activity, with an SI of > 100-fold greater toxicity towards one or both of the P. falciparum clones (D6 and W2), relative to KB cells ¹⁵.

In animal models of malaria, large experimental variability of the results is associated with drug, parasite and host interactions. For ethical reasons, the number of animals used may not be increased to more accurately characterize the antiparasitic effects. Despite this low experimental reproducibility, the murine malaria model used herein is an important tool in anti-malarial drug discovery and development programs ²⁰. The 4-day suppressive test, which primarily evaluates the antimalarial activity of candidate agents in early infections, and the prophylactic test, which evaluates the capability of candidate extracts in established infections, is commonly used for antimalarial drug screening. In both methods, the determination of the percentage of inhibition of parasitemia is the most reliable parameter.

TABLE 2: SUPPRESSIVE ACTIVITIES OF FRACTION ALKALOID OF M. CHARANTIA FRUITS DURING EARLY P. BERGHEI BERGHEI NK 65 INFECTION IN MICE (4-DAY TEST)

Data are expressed as mean ± SEM for five animals per group^{; *}P< 0.05 when compared with control

TABLE 3: PROPHYLACTIC EFFECT OF FRACTION ALKALOID OF M. CHARANTIA FRUIT ON PARASITAEMIA IN MICE

The alkaloid fraction of M. charantia fruit exerted a dose-dependent chemo-suppressive effect against P. berghei berghei NK 65. The fraction gave a significant (P<0.05) chemo suppression of 37% for the low dose of 1 mg/kg and 74% for the highest dose of 100 mg/kg when compared to the control. The observed higher efficacy of the standard drug, chloroquine (84%), which was higher than fraction treated groups, may in part be due to nonselectivity of the fraction or slow absorption and poor bioavailability of the fraction. In the prophylactic study, the alkaloid fraction of M. charantia fruit significantly (P<0.05) exerted a dose-dependent reduction in the level of parasitemia in the fraction treated groups, while the standard drug chloroquine gives the highest effect. As shown in Table 3, prophylactic activities were slightly lower than the suppressive activities for alkaloid rich-fraction. At 100 mg/kg/day, the alkaloid rich-fraction, M. charantia, exhibited a high (73%) parasite reduction. Chloroquine (10 mg/kg/day) was used as reference drugs and showed a parasite reduction of 90%.

The prophylactic effect of the alkaloid fraction M. charantia had shown antiplasmodial activity against P. berghei infected mice with maximum parasitemia chemo suppression of 74% at a dose of 100 mg/kg body weight dose. Unlike the 4-day suppression, chloroquine 5 mg/kg body weight did not show 100% parasitemia eradication; rather, it inhibited 90% Table 2. The high parasite count in the prophylactic test both in the alkaloid fraction M. charantia and in reference standard drugs treated groups might be attributed to the rapid metabolism of the administered alkaloid fraction M. charantia and reference standard drugs (before inoculation) to inactive products where the fraction and reference standard drugs were initially administered for four days (prophylactic test) before inoculation with P. berghei parasite ²¹.

The zebrafish is a vertebrate animal model that is increasingly used for in-vivo drug toxicity and efficacy screening, and for assessing chemical toxicity and safety ^{22, 23}. In contrast to other vertebrate models, the zebrafish completes embryogenesis in the first 72 h. Advantages of the zebrafish embryotoxicity test include the detailed knowledge available about embryogenesis in zebrafish and the independence of embryo development from maternal fish. Also, transparency of embryos makes it easy to monitor and evaluate. Furthermore, development takes only three days from fertilization until hatching, and at these stages, the zebrafish embryo is not considered a laboratory animal under European legislation. These advantages make the zebrafish embryo model suitable for relatively high-throughput tests ²⁴.

FIG. 1: MORTALITY OF D. RERIO EMBRYOS TREATED WITH ALKALOID RICH- FRACTION M. CHARANTIA FRUIT AT 96 h POST TREATMENT APPLICATION. THE MORTALITY OF EMBRYOS TREATED WITH 600 PPM OR HIGHER OF FRACTION WAS SIGNIFICANTLY HIGHER THAN THAT OF THE CONTROL EMBRYOS

Embryo-toxicity using D. rerio as a model is an important element for assessing the adverse effects of certain substance on cell structures and processes that are intrinsic to almost all cells. The toxic effect of the alkaloid fraction M. charantia fruit on D. rerio embryos was recorded, and the mean percentage mortality of embryos after 96 h of treatment exposure is shown in Fig. 1. It can be seen that the toxic effect of the alkaloid fraction M. charantia fruit was concentration-dependent, as evidenced by the increased mortality of embryos with a higher concentration of extract.

Fig. 1 shows alkaloid rich-fraction of bitter melon (M. charantia) fruits with high concentration (600-900 ppm). At a concentration of 600 ppm, embryos that hatched were only 65%; even at a concentration of 900 ppm, no embryo was hatched. Failure for the embryo to hatch might lead to the embryos’ mortality. The highest mortality rate was seen at the concentrations of 700 ppm (60%), 800 ppm (80%), and 900 ppm (100%). Table 4 and Fig. 2 present the teratogenic effects on D. rerio embryo due to alkaloid rich-fraction exposure.

TABLE 4: TERATOGENIC EFFECT OF ALKALOID RICH FRACTION EXPOSURE ON ZEBRA FISH EMBRYO

^a Number of embryos exposed to teratogenic effects at all concentrations and all treatment periods; ^b, Number of embryos exposed to teratogenic effects /number of abnormal embryos at all concentrations and all treatment periods; ^*, Major malformations (>50%). An embryo may have more than one malformation.

Administration of alkaloid rich-fraction of M. charantia fruits induced teratogenic effects in the organs of the zebrafish embryo. This was evident with the malformation seen in the embryo. Mal-formation refers to the abnormal growth of an organ or tissue. Abnormality was seen in the growth of pericardial area (51.28%) and yolk sac (51.28%), both of which were major malformations 50%, while the growth in the axial area (10.26%) and circulation (30.77%) were minor malformations. Major malformation began to be seen at a concentration of 600 to 800 ppm since 24 jpf.

FIG. 2: MORPHOLOGY OF NORMAL ZEBRAFISH EMBRYO IN CONTROL GROUP, AND SOME ABNORMALITIES IN TREATMENT GROUP WITH ALKALOID FRACTION: (A) NORMAL EMBRYO K- 24 HPF, (B) K-48, (C) PERICARDIAL EDEMA (72 HPF, 800 PPM), (D) EMBRYO’S COAGULATION (96 HPF, 800 PPM), (E) PERICARDIAL EDEMA AND YOLK EDEMA (72 HPF, 800 PPM), (F) TAIL MALFORMATION (72 HPF, 800 PPM)

The present study provides the first examination of observable alkaloid fraction of bitter melon (M. charantia) toxicity on zebrafish development. Morphological developments such as the tail, head, embryonic movement, pigmentation, heart, yolk sac, and hatching were within the normal range when compared to the normal group. From the acute toxicity test, it was observed that the fraction alkaloid of M. charantia fish mortality rate increased with increasing concentration of the fraction alkaloid.

The zebrafish embryos that were exposed to sublethal concentrations of these extracts showed some developmental abnormalities. Most of the fraction alkaloid M. charantia induced abnormalities in developing heart and yolk sac in a dose-dependent manner. The embryos exposed to the extracts had cardiac edema with an enlarged cardiac chamber (cardiac hypertrophy) compared with the control. The major alkaloid in the betel nut, arecholine treated embryos, exhibited general developmental retardation in a dose-dependent manner by depletion of intracellular thiols; Birth Defects Research ²³.

CONCLUSION: The result of this study showed that the fraction-rich alkaloid of bitter melon (M. charantia) have antimalarial potentials; therefore, the plant should be extensively investigated to isolate and identify their active antimalarial constituents.

This study also concludes that alkaloid fraction may lead to abnormal development of the heart and yolk sac in zebrafish.

ACKNOWLEDGEMENT: Our gratitude goes to the Ministry of Research, Technology and Higher Education Indonesia that has funded this research under Competence Grant program.

CONFLICT OF INTEREST: There are no conflicts of interest.

REFERENCES:

1. WHO: Global technical strategy for malaria 2016 - 2030. Global Malaria Programme Publisher 2015. http.who.int/malaria/publications/atoz/
2. Ministry of health Republic of Indonesia: Indonesia Health Profile 2013. http://www.kemkes.go.id.
3. Mita T, Tanabe K and Kita K: Spread and evolution of Plasmodium falciparum drug resistance. Parasitol Int 2009; 59(3): 201-09.
4. Saxena S, Pant N, Jain DC and Bhaluni RS: Antimalarial agents from plant sources. Curr Sci 2003; 85: 1314-29.
5. Syamsudin, Risma MT, Yanti S and Yunahara F: Ethno-botanical survey of plants used in the traditional treatment of malaria in Sei Kepayang, Asahan, North Sumatera. Asian Pac J Trop Med 2014; 7(1): 104-07.
6. Ameyaw Y and Duker-Eshun G: The alkaloid contents of the ethno-plant organs of three antimalarial medicinal plant species in the eastern region of Ghana. Int J Chem Sci 2009; 7(1): 48-58.
7. Trager W and Jensen JB: Human malaria parasites in continuous culture. Science 1976; 193(4254): 673-75.
8. Kraft C, Jenett-Siems K, Siems K, Jakupovic J, Mavi S, Bienzle U and Eich E: In-vitro antiplasmodial evaluation of medicinal plants from Zimbabwe. Phytother Res 2003; 17: 123-28.
9. Peters W, Portus JH and Robinson BL: The chemotherapy of rodent malaria. XXII. The value of drug-resistant strains of berghei in screening for blood schizonticidal activity. Ann Trop Med Parasitol 1975; 69: 155-71.
10. Waako B, Gumede P, Smith and Folb PI: The in-vitro and in-vivo antimalarial activity of halicacabum L. and M. foetida Schumch. Et Thonn J Ethnopharmacol 2005; 99(1): 137-43.
11. Dulay RMR, Kalaw SP, Reyes RG, Alfonso NF and Eguchi F: Teratogenic and toxic effects of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W. Curt.: Fr.) P. Karst (higher basidiomycetes), on zebrafish embryo as model. International Journal of Medicinal Mushrooms 2012; 14(5): 507-12.
12. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B and Schilling TF: Stages of embryonic development of the zebrafish. Dev Dyn 1995; 203(3): 253-10.
13. Brandhof L, van der Ven TM and Aldert HP: Concentration-response analysis of differential gene expression in the zebrafish embryotoxicity test following flusilazole exposure. Toxicol Sci 2012; 127(1): 303-312.
14. Angerhofer CK, Guinaudeau H, Wongpanich V, Pezzuto JM and Cordell GA: Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J Nat Prod 1999; 62: 59-66.
15. Mueller MS, Karhagomba IB, Hirt HM and Wemakor E: The potential of Artemisia annua as a locally produced remedy for malaria in the tropics: agricultural, chemical and clinical aspects. J Ethnopharm 2000; 73(3): 487-93.
16. Syamsudin, Tambunan RM, Farida Y, Sandhiutami NMD, and Dewi RM: Phytochemical screening and antimalarial activity of some plants traditionally used in Indonesia. Asian Pac J TropDis 2015; 5(6): 454-57.
17. Yousif SA: In-vitro screening of antiplasmodium activity of Momordica charantia. J Nat Resour Environ Stud 2014; 2(3): 29-30.
18. Tackie T and Schiff Jnr PL: Cryptospirolepine: A unique spiro-noncyclic alkaloid isolated from Cryptolepis sanguinolenta. J Nat Prod 1993; 56: 653-55.
19. Renata BS, Rocha e Silva L, Melo MR, Costa JS, Picanco NS and Lima SW: In-vitro and in-vivo anti-malarial activity of plants from the Brazilian Amazon. Malar J 2015; 14: 508.
20. Dahanukar SA, Kulkarni FA and Rege NN: Pharmacology of medicinal plants and natural products. Indian J Pharmacol 2000; 32: 1081-118.
21. Ali S, Champagne DL, Spaink HP and Richardson MK: Zebrafish embryos and larvae: a new generation of disease models and drug screens. Birth Defects Res C Embryo Today 2011; 93(2): 115-33.
22. McGrath P and Li C: Zebrafish: A predictive model for assessing drug-induced toxicity. Drug Discov Today 2008; 13(9-10): 394-01.
23. Chang BE, Liao MH, Kuo MY and Chen CH: Developmental toxicity of arecoline, the major alkaloid in betel nuts, in zebrafish embryos. Clin Molec Teratol 2004; 70(1): 28-36.
    

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    How to cite this article:

    Abdillah S, Farida Y, Kartiningsih, Sandhiutami NMD and Mohamad K: Antimalarial activity and toxicity evaluation of the alkaloid-rich fraction of Momordica charantia fruits. Int J Pharm Sci & Res 2019; 10(5): 2516-22. doi: 10.13040/IJPSR.0975-8232.10(5).2516-22.

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