Chemical Composition and In Vitro Antiplasmodial Activity of the Total Alkaloids of the Bulbs of Two Amaryllidaceae Species from Northern Peru

of the Total


INTRODUCTION
Malaria is an infectious and life-threatening disease caused by Plasmodium parasites such as Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax and Plasmodium malariae; Among these protozoa, P. falciparum is believed to be responsible for most serious diseases and most fatal cases 1 .In 2018, the World Health Organization (WHO) declared 228 million cases of malaria worldwide; mostly in the African Region, followed by the Eastern Mediterranean, Western Pacific and Southeast Asia; including 405,000 deaths 2 .To face this situation, WHO has recommended the use of therapies based on combinations of artemisinin and derivatives with other drugs; however, in some countries P. falciparum is already resistant to artemisinin combination therapies 3 .
The Global Technical Strategy for Malaria 2016-2030 defined the goal for 2030 to decrease the 90% of the incidence rate of malaria as well as related death rate 4 .To contribute with this purpose, we need to find new sources of medicaments against this illness.In this sense, plants are the main source of medicinal agents, even a large part of the world's population use herbs and it is not surprising to find a well-established system of traditional medicine in many countries 5 .The recognition and validation of traditional medicine is important and could lead to the discovery of new plant-derived drugs, such as quinine isolated from Cinchona species and artemisinin isolated from Artemisia annua L 6 .In addition, many compounds from various medicinal plants were isolated and showed in vitro and in vivo antiplasmodial activity against 7 .
The species Clinanthus incarnatus and Clinanthus ruber belong to the Amaryllidaceae family.This monocotyledonous botanical family is widely distributed throughout the world with approximately 70 genera and 1600 species.Besides there are 28 genera in South America and 24 genera in Peru, finding in this territory 138 species, among which are 15 to 20 species belonging to genus Clinanthus 8 .Amaryllidaceae family plants contain, especially in the bulbs, a variety of unique alkaloids not present in other families.These isoquinoline alkaloids have powerful medicinal properties, including antitumor, antiviral, cytotoxic, acetylcholinesterase inhibitory, immunostimulating, anti-inflammatory, analgesic, and for the treatment of Alzheimer's disease 9 .Some of these alkaloids are of particular interest due to their potential antiprotozoal activity such as lycorine, agustinine and crinamine from Crinum amabile bulb, in addition, Haemanthamine and 6-hydroxyhaemanthamine exhibited antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum 10 .was to determine the chemical composition and evaluate the in vitro antiplasmodial activity of the total alkaloids of the bulbs of two amaryllidaceae species from northern Peru.

Extraction of alkaloids
The bulbs were washed, disinfected and cut into thin slices.Then they were dried in a forced convection oven at 40 ºC for 72 hours.Once the plant material was completely dried, it was ground in a rotary blade mill.The dried powdered material was macerated with methanol for 72 hours at room temperature, applying ultrasonic baths at intervals of 1 to 2 hours.Subsequently, the methanolic extract was filtered and evaporated to dryness under reduced pressure using a rotary evaporator at a temperature of 40 °C.The crude extract obtained was subjected to acidification with H 2 SO 4 (2% v/v) and was cleaned with ethyl ether, separating the organic phase composed of neutral materials such as chlorophylls, waxes and mucilages from the aqueous phase, rich in alkaloids.The acidic aqueous phase was subjected to basification with NH 4 OH (10% v/v) until reaching a pH of 10, then the alkaloids were extracted through the repeated use of chloroform, so that the alkaloids were retained in the organic phase.Then the solvent was evaporated under reduced pressure in the rotary evaporator at a temperature of 45 °C, obtaining the extract of total alkaloids (TA) [11][12][13] .

GC-MS conditions
Alkaloids were identified by using a GC-MS apparatus (Agilent Technologies 6890 N coupled with MSD5975 inert XL) operating in the electron ionization (EI) mode at 70 eV.A Sapiens-X5 MS column (30 m x 0.25 mm i.d., film thickness 0.25 µm) was used.The temperature gradient was as follows: 12 min at 100 °C, 100-180 °C at 15 °C/min, 180-300 °C at 5 °C/min and 10 min hold at 300 °C.The injector and detector temperatures were 250 and 280 °C, respectively, and the flowrate of carrier gas (He) was 1 ml/min.Two mg of each total alkaloids was dissolved in 1 ml of MeOH: CHCl 3 (1:1, v/v) and 1 µl was injected using the split-less mode.Codeine (50 µg/ml) was used as an internal standard.

Alkaloid quantification
To quantify the single constituents, a calibration curve of galanthamine (10, 20, 40, 60, 80 and 100 µg/ml) was used.The same amount of codeine (50 µg/ml) was added to each sample as an internal standard.The peak areas were manually obtained considering selected ions for each compound (base peak of their MS, i.e., m/z at 286 for galanthamine and 299 for codeine).The ratio between values obtained for galanthamine and codeine in each solution was plotted against the corresponding concentration of galanthamine to obtain the calibration curve and its equation (y = 0.0224x-0.2037;R2 = 0.9977).All data was standardized to the internal standard area (codeine) and the equation obtained for the calibration curve of galanthamine (GAL) was used to calculate the amount of each alkaloid.Results are expressed as µg GAL/ 100 mg TA (total alkaloid).

In vitro antiplasmodial activity
The evaluation of the antiplasmodial activity of the total alkaloids was carried out in vitro with strain FCR3 (chloroquine resistant) of Plasmodium falciparum, which were cultured in RPMI 1640 medium supplemented with 10% human serum and a hematocrit of 4% that was obtained adding 200 µl of total red blood cells in 4.5 ml of RPMI 1640 and 0.5 ml of serum or plasma (Blood group 0, Rh + ) and incubated at 37 o C in a 5% O 2 6% gas mixture atmosphere of CO 2 and balanced N 2 , as described by Trager W, et al, with some modifications.The tests for the antiplasmodial activity of the total alkaloids (1.0, 2.5, 5.0, 10.0, 25.0 and 50.0 µg /ml dissolved in DMSO), were carried out in 96-well plates of flat bottom, for each alkaloidal extract in triplicate.Chloroquine diphosphate (10 to 1000 nM) was used as a control of the test.The cultures were synchronized with a parasitaemia and a hematocrit of 1 and 2% respectively; These were dispensed in a volume of 100 µl in 96-well plates in duplicate, 100 µl of the total alkaloids were added, and finally they were incubated at 37 ° C for 48 hours.After this incubation time, the upper phase of the culture was completely eliminated, to make a smear of the sediment from each well, then fixing with methanol and staining with Giemsa.These plates were observed under the microscope with a 100x immersion lens, counting uninfected red blood cells (GRL) and infected red blood cells (GRI), to obtain the percentage (%) of Inhibition calculated by the formula [14][15][16] : The IC 50 value was calculated by an activity curve: Percentage of inhibition vs. logarithm of drug concentration, through linear interpolation calculation:

Statistic analysis
The results were processed using the statistical program SPSS v. 23 and, expressed as the arithmetic median ± standard deviation.The relationship between the groups was determined using the one-way ANOVA test, in which p0.05 were considered statistically significant.

Alkaloids Identified in C. incarnatus and C. ruber by GC-MS
The identified alkaloids and their structures are represented in Table 1 and Figure1.The alkaloids present in the analyzed samples were identified by comparing their GC-MS spectra and Kovats retention index (RI) values with those of authentic Amaryllidaceae alkaloids previously isolated and identified by spectrometric methods (NMR, UV, CD, IR, MS) in the Natural Products Laboratory of Barcelona University, the NIST 05 Database, or literature data.The MS spectra were deconvoluted by AMDIS 2.64 software (NIST).
Shows that there is a directly proportional relationship between the concentration of total alkaloids and the percentage of inhibition of the P. falciparum with values of 23.5 ± 0.46% at 90.1 ± 0.1% (C.incarnatus) and 31.5 ± 0.1% at 94 ± 0.56% (C.ruber).Besides, in figure 2, the values of IC 50 0.375 μg/ml (C.incarnatus) and IC 50 0.241 μg/ml (C.ruber) are shown, indicating antiplasmodial activity.ANOVA and Tukey test were applied, determining that there is a statistically significant difference between the percentages of inhibition and IC 50 values between both species and chloroquine (p <0.05) The data presented correspond to the average of three replications ± standard deviation.The asterisks represent a significant difference in relation to the control (chloroquine) according to the ANOVA test (p <0.05).

DISCUSSION
The amaryllidaceae family is distinguished for its exclusive alkaloids isolated from all its genera 17 .Generally are isoquinoline type alkaloids that have not been identified in any other plant family and are classified into nine different types based on the heterocyclic system: norbelladine, lycorine, homolycorine, crinine, hemantamine, narcyclisine, tazetine, montanine and galantamine 18,19.In this sense, the alkaloids found in both species in this research belong to lycorine type as well as crinine/haemanthamine-type, founding that lycorine is the majority alkaloid in both species with values of 19.73 µg GAL / 100 mg TA ( 0.01973%) and 70.2 µg GAL /100 mg TA (0.0702%) respectively; what matches other investigations in amaryllidaceae species where concentrations ranged from 0.006% to 0.162% for Galanthus elwesii 20 , 0.10-0.53%for Sternbergia sicula 21 , 0.19-0.40%for Sternbergia lutea 21 and 0.05-0.14%for Pancratium maritimum 21 , 0.009% to 0.012% for Galanthus trojanus) 22 , and 0.004% for Galanthus cilicicus 22 .It should be noted that the variability in the concentration depends on the environmental conditions and stress factors to which the plant is exposed [20][21][22] .Besides lycorine has a variety of biological activities (antineoplastic, immunostimulant, bacteriostatic, anticholinesterase, analgesic, anti-inflammatory, antiviral, antiprotozoal and antimalarial) 23 .
The bulbs of Clinanthus incarnatus (Kunth) Meerow and Clinanthus ruber (Herb.)Meerow & A. Cano were collected from the districts of Otuzco (2641masl) and Pataz (3118 masl) in La Libertad Region.The botanical identification was carried out by Dr. Alan Meerow from Agricultural Research Service, United State Department of Agriculture, Miami, FL (USA), and deposited in the Herbarium Truxillense of the National University of Trujillo (HUT).

Figure 1 :
Figure 1: In vitro antiplasmodial activity of the total alkaloids of the bulbs of Clinanthus incarnatus and Clinanthus ruber.

Figure 2 :
Figure 2: CI 50 of the total alkaloids (TA) of the bulbs of Clinanthus incarnatus and Clinanthus ruber and cloroquine.

Table 1 : Alkaloids identified in Clinanthus incarnatus and Clinanthus ruber by GC-MS. Values in µg GAL/100 mg TA.
* proposed structure-type according to the fragmentation pattern; Rt: retention time; RI:Kovats Retention Index; NI: not identified.