Background
Throughout the history of mankind, malaria has been one of the major causes of human illness and death. More than 800,000 deaths occur every year; the vast majority being children under the age of five. Thus this highly infectious disease has a global impact. Malaria is a parasitic disease widespread in tropical and subtropical regions of the world [
1,
2]. It is endemic particularly in regions of Africa, Asia and South America. India’s extensive geography and diverse climate supports ideal environments for sustaining malarial parasites and their vectors [
3]. Malaria can be diagnosed easily on morphological basis at different stages of parasite in human blood; with the exception of
P. falciparum. [
4].
P. falciparum is the most severe strain of the malaria due to highest human deaths and resistant to standard antimalarial drugs. [
5]. The WHO has recommended artemisinin-based combination therapy (ACT) as the first line treatment for multidrug resistant malaria caused by
P. falciparum in different parts of the word [
6]. Recent studies have reported that
P. falciparum has developed resistance to many of available antimalarial drugs [
7‐
9].
Malaria has become a leading cause of morbidity and mortality mainly due to its prevalence in poor resource countries; where the therapy is unaffordable due to non-availability of oral administered drugs [
10]. As antimalarial drug resistance is undermining the effective treatment of the disease; there is a critical need for effective, safe, and affordable antimalarial agents. Herbal medicine occupies a pivotal role in treating infectious diseases since onset of mankind. It is estimated that about 40% of all medicines is either natural products or their semi-synthetic derivatives [
11].
Natural products may offer relatively cheap alternative treatment opportunities for malaria patients due to vast metabolic diversity [
12‐
14]. Currently used antimalarial drugs such as quinine and artemisinin were both isolated from plants
Cinchona officinalis
and
Artemisia annua respectively. Consequently, it has been established that plants have potential as sources for antimalarial drugs. The diverse climates of India flourish huge diversity of medicinal plants and use of plants for treating ailments tracks back to ancient Indian [
15].
Quality evaluation and pharmacological standardization of herbal preparation is a fundamental requirement of industry for commercial production. According to WHO guidelines, an herbal product needs to be standardized with respect to safety before releasing it into the market [
16]. HPTLC (high performance thin layer chromatography) is an inexpensive method for separation, qualitative identification, or semi-quantitative analysis of samples and it can be used to solve many qualitative and quantitative analytical problems in a wide range of fields; including medicine, pharmaceuticals, chemistry, biochemistry, food analysis, toxicology and environmental analysis [
17].
Aloe vera (syn.:
Aloe barbadensis Miller) is the most commercialized
Aloe species belonging to the Xanthorrhoeaceae family [
18,
19]. There are many natural medicinal herbs, but
Aloe vera possesses a vast array of healing benefits. Owing to its multipurpose utility,
Aloe has been introduced into cultivation as a household plant. It has been in use since ages as folk medicine.
Aloe vera is a rich source of over 200 naturally occurring nutrients which contain water soluble and fat soluble vitamins, minerals, enzymes, polysaccharides, phenolic compounds and organic acids [
20]. Its secondary metabolites have multiple properties such as anti-inflammatory, antibacterial, antioxidant, immune boosting, anticancer, antiageing, sunburn relief and antidiabetic potentials [
21‐
23]. Several traditional uses also have been reported such as burn injury, eczema, cosmetics, inflammation, and fever [
24].
Aloe juice mixed with water and honey is used as an effective antimalarial cure in Yemen [
25].
Recently
Aloe vera was reported to be used against malaria parasite with the highest frequency in a documentation report on medicinal plants used by the local communities of western Uganda [
26]. Van Zyl and Viljoen [
27] screened the main constituents of 34
Aloe species for antiplasmodial activity using the titrated hypoxanthine incorporation assay. They have observed that methanol extracts possessed antiplasmodial activity against
Plasmodium falciparum strain at concentration ranged from 32 to 77 μg ml
−1 where 50% of the parasite growth was inhibited (IC
50 value) [
27]. The Aloe species of
A. secundiflora and
A. lateritia were also used for treating malaria and related symptoms [
28].
Geographical conditions are the main factors that ultimately affect the phytoconstituents and medicinal properties of a plant. India has six major climatic zones: Highland, semi-arid, arid, tropical wet, tropical wet and dry, and humid subtropical climate.
Aloe vera grows all over the India, wildly in Maharashtra and Tamil Nadu states where as Andhra Pradesh, Gujarat and Rajasthan states are known for its cultivation. [
29]. So, keeping in view of the importance of
Aloe vera plant the present work is an attempt to evaluate the antiplasmodial activity and phytochemical standardization of
Aloe vera aqueous extracts with HPTLC; collected from different climatic regions of India to elaborate the effect of climatic conditions on phytochemical diversity and activity of samples.
Discussion
According to the WHO report (2016), there were an estimated 429,000 malaria deaths and 212 million new cases of malaria reported worldwide. This report revealed that 778,821 cases have been recorded with
P. falciparum and 390,440 cases with
P. vivax causing malarial infections in India. The WHO malaria report stated that African Region accounted for most global cases of malaria (90%), followed by the South-East Asia Region (7%) and the Eastern Mediterranean Region (2%) [
5]. The 22% of India’s population live in high transmission, 67% live in low transmission areas and 11% live in malaria-free areas. The incidence of malaria in India accounted for 58% of cases in the South East Asia Region [
33]. The central and eastern regions of India were reported for the most malaria cases particularly the eastern states of Odisha, West Bengal, and Jharkhand, the central states of Chhattisgarh and Madhya Pradesh, and the western states of Gujarat, Karnataka and Rajasthan, with the largest number of deaths reported in Odisha [
34]. The diverse malaria epidemiology in India is mirrored by high diversity of malaria vector species, most of which exist as complexes comprising several cryptic species that vary in vectorial capacity [
35,
36].
The climate varies from tropical monsoon in the south of the country to temperate in the north. Such climatic variation is due to a sharp temperature gradient caused by atmospheric changes in wind circulation and precipitation, lending to seasonally dependent asymmetric heating patterns of India’s peripheral bodies of water and land [
3]. The new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance. The regions and periods of the year of plant collection are known to play an important role in the variation of the type of compounds found in plants as well as their concentration. Geographical conditions are the main factors for genetic diversity that ultimately affect the phytoconstituents and medicinal properties of a plant [
37]. Diversity among organisms is a result of variations in DNA sequences and environmental effects. Species with a wide geographic area generally have more genetic diversity [
38]. According to previous findings it is suggested that environmental temperature has a significant effect on antioxidant activity evaluation and it is more pronounced in cold weather [
39]. This promoted us to collect samples in the winter season (Jan- Feb 2013).
Aloe vera grows all over the India and has a wide range of bio- active constituents found in leaves [
40]. Nine categories of phytochemical constituents of
Aloe vera can be classified as, anthraquinones
, inorganic compounds, amino acids, fatty acids, alkaloids, carbohydrates, enzymes, and vitamins along with other miscellaneous compounds [
41]. Anthraquinones are the most important active ingredients of
Aloe vera [
42]. The antiplasmodial activity of
A. vera may be explained in the light of the presence of anthraquinones and other quinoid compounds which exert good activity against
P. falciparum [
43]. The four main anthraquinones showing quite high medical values are aloin, aloe-emodin, aloe bitter and aloe lectin [
44].
Phytochemical extraction from plant samples greatly relies upon solvent and extract preparation methods [
45,
46]. Water is the most polar of the solvents and confers negligible toxicity as such was used in this study [
47,
48]. Standardization of natural products is a complex task due to their heterogeneous composition, which is in the form of whole plant, plant parts, or extracts obtained thereof. To ensure reproducible quality of herbal products, proper control of starting material is of utmost importance. For identification of the crude drug it is best to possess the authentic reference standard of that particular crude drug and HPTLC is a valuable tool for the investigation of herbal products with respect to different aspects of their quality and quantity [
49,
50].
In present study, HPTLC analysis showed the variability in the amount of aloin and aloe-emodin anthraquinones contents with respect to diverse climatic conditions of Indian agro- climatic zones. The quantity of aloin is found higher in comparison to aloe-emodin. The previous studies also revealed the higher amount of aloin compare to aloe-emodin [
51‐
53]. Maximum amount of aloin was detected in Jammu and Kashmir and Himachal Pradesh (Highland zone, with average temperature 13.5 °C and average rainfall 1011 mm and 1251 mm respectively) samples where as that of aloe-emodin in Punjab (Semi-arid zone, with average temperature of 25 °C and average rainfall 649 mm) sample.
There is not too much literature available on evaluation of antiplasmodial potential of
Aloe vera but the plant has been reported several times as a antimalarial remedy in folk medicine system [
25,
26]. Efforts are now being directed towards the discovery and development of new chemically diverse antimalarial agents. The South African
Vitex spp. showed significant activity against
P. falciparum chloroquine-resistant FCR-3, with IC
50 values ranging from 9.16 ± 1.37 μg/ml to 16.02 ± 3.07 μg/ml. [
54]. The plant extracts of 134 species tested for in vitro activity against a
Plasmodium falciparum strain D10 using the parasite lactate dehydrogenase (pLDH) assay showed the 49% promising antiplasmodial activity (IC
50 ≤ 10 g/ml), while 17% were found to be highly active (IC
50 ≤ 5 g/ml) [
55].
Larvicidal activity of
Aloe species growing in Kenya, namely
Aloe turkanensis,
Aloe ngongensis and
Aloe fibrosa tested against
A. gambie third instar larvae showed 60% mortality at concentration of 2 mg/ml or 0.2%
w/
v [
56]. Several anthraquinones extracted from different plant parts strongly inhibited in vitro growth of a chloroquine sensitive strain of
Plasmodium falciparum (3D7) [
57]. Anthraquinones may generate reactive oxygen and thus inactivate malaria parasites (
Plasmodium falciparum, P. vinckei and P. berghei) [
58,
59]. Latex leaf extract of
Aloe citrine showed antiplasmodial activity due to presence of anthrone, homonataloin A/B as a major constituent [
60]. On the basis of our present findings, Himachal Pradesh sample showed the lowest EC
50 value (0.289) which means highest antiplasmodial activity in comparison to other
Aloe vera samples.
Aloe vera is a cold sensitive plant. During stress more phytochemicals are produced in plants to withstand the adverse conditions. Studies conducted on plants in stress conditions showed higher production of flavonoids, anthocyanins and mucilaginous substances [
53].
In the present study EC
50 values of aloin and aloe-emodin were 67 μg/ml and 22 μg/ml respectively for antiplasmodial activity against a chloroquine (CQ)-sensitive strain of
P. falciparum (MRC-2). As both the anthraquinones tested in the study showed less antiplasmodial potential in comparison to crude extracts of different
Aloe vera samples, these data suggest that the activity observed may be due to the synergistic effect of aloin, aloe-emodin and presence of other more active compounds in the extracts of
Aloe vera plant. In contrast the previous study on Aloe extracts revealed that IC
50 value for aloin was 169.76 ± 11.5 μg/ml against the chloroquine-sensitive 3D7 strain [
27]. Dai et al. [
61] reported the IC
50 value for antiplasmodial activity of aloe-emodin was 50 μg/ml isolated from ethanol extract of South African plant
Kniphofia ensifolia against
P. falciparum Dd2 strain. They also showed that esterification of the primary hydroxyl group of aloe-emodin with various carboxylic acids increased its antiplasmodial activity, with the most potent analogue being the 3,4-dimethylcaffeic acid derivative with an IC
50 value of 1.3 ± 0.2 μM) over 40 times than that of aloe-emodin. The biological activities of
Aloe vera are due to the synergistic action of a variety of compounds, rather than from a single defined component [
62,
63]. Significant correlation between quantities of both the anthraquinones used as marker compounds and EC
50 values of the different
Aloe vera extracts proves the plant as a prospective antimalarial remedy.
Traditional medicine and ethnobotanical information play an important role today as subject for scientific research, particularly when the literature and field work data have been properly evaluated. From the present work, it may be concluded that agro-climatic locations along with temperature and rainfall have significant effects on the
Aloe vera plant phytoconstituents and its antimalarial potential. However, there is still a need to investigate the effects of different biotic and abiotic factors on
Aloe vera. In future, it is also required to isolate antiplasmodial molecules from crude extracts of
Aloe vera to enhance the antimalarial potential of the plant under cold stress. Plant growth and productivity are greatly affected by environmental stresses such as dehydration, high salinity, low temperature and biotic pathogen infection. Many plant genes are regulated in response to biotic and abiotic stresses and their gene products function in stress response. Such genetic systems are thought to be very important in increasing tolerance of plants to these stresses as well as in management for successful crop cultivation [
64].