Phytochemical Screening and in vitro Antimicrobial Activity of Waltheria indica Linn Root Extracts

The aim of this study was to examine the phytochemical constituents and evaluate the antimicrobial activity of Waltherica indica Linn root extracts in solvents of different polarities (ethyl acetate, chloroform and chloroform: methanol (1:1). The experiment was conducted at the Chemistry Advanced Research Centre of Sheda Science and Technology Complex, Abuja, Nigeria between November 2016 and January 2017. Clinical strains of five bacteria and three fungi isolates were utilized. Chloroform, ethyl acetate and chloroform: methanol (1:1) root extracts were tested at 12.5, 25.0 and 50.0 μg/ml using the Agar well diffusion technique. The minimum inhibitory, minimum bactericidal and minimum fungicidal concentrations of each solvent extract were assessed. Phytochemical analysis was also performed using ethanol as the extraction solvent. The phytochemical compounds obtained in the methanol extracts where alkaloids, cardiac glycosides, flavanoids, phenolic acids, saponins, steroids, tannins and terpenoids. Agar well diffusion used for sensitivity study of S. aureus, S. pneumonia, S. pyrogens, K. pneumonia, C. ulcerans, C. albicans, C. krusei and C. tropicalis with 10μg/ml ciprofloxacin and 30μg/ml fluconazole revealed that the ethyl acetate extract of Waltheria indica Linn gave 27±0.20 to 31±0.95mm zones of inhibition, whereas the chloroform extract gave an inhibitory range of 26±0.15 to 30±0.25mm while the chloroform: methanol extracts gave a 26±0.35 to 29±0.85mm zones of inhibition respectively in comparison to the 31±0.65 to 36±0.35mm obtained from the control. The minimal inhibitory content (MIC) of the ethyl acetate extracts was recorded at 0.25 mg/ml, while the MIC values for the chloroform and chloroform: methanol extracts were between 0.25 and 0.50 mg/ml respectively. The results obtained suggested that the studied plants possess anti-microbial spectrum aligned to the phyto-constituents.


Introduction
Plants indigenous to West African possess a rich array of bioactive compounds, useful for the treatment of a variety of diseases that are unique to the region [1]. Traditional medicine, practiced across the African continent, utilize a large number of plants whose diversity creates an arsenal of multifunctional phyto-compounds. These phyto-compounds serve as essential raw materials for new drug discovery whereby they possess the inherent advantage of serving as a cheaper, readily available therapeutic agent. For example, Artemisia annua, indigenous to the African region, was discovered to be active against Plasmodium falciparum through a series of in vitro experiments, the result of which lead to the isolation of artemisinin towards the treatment of malaria [2]. Other plants like Lunasia amara and Solanum sodomaeum where found to be active against Mycobacterium tuberculosis, thus prompting the isolation of Graveolinine and Solsodomine respectively for the treatment of tuberculosis [3].
Nigeria is one of several countries in the African country that possesses a rich diversity of untapped botanical resources which could be applied to the treatment of many diseases prone to the country [1,4]. One of such plants is Waltheria indica Linn (hankufah in Hausa, korikodi in Yoruba), widely distributed across the South Western and North Central parts of Nigeria. Traditional uses of the plant includes the treatment of diarrhea, stomach ache, wounds, cough, haemorrhage, fever, malaria, skin diseases and can be consumed as an aphrodisiac [6,7]. With the known uses of the plant, it is possible that Waltheria indica Linn can be utilized in the treatment of other medical conditions brought about by pathogenic organisms. Diseases causing pathogens that have commonly been isolated from community and hospital acquired upper respiratory tract infections include Staphylococcus aureus, Streptococcus pneumonia, Klebsciella pneumonia, Candida spp., etc [8]. As part of our research efforts on Nigerian medicinal plant parts, this study was conducted to examine the phytochemical constituents and determine the antimicrobial activity of the roots of Waltheria indica Linn.

Materials and Methods
The roots of Waltheria indica Linn were collected in fresh condition from Kwali (8°50'49''N 7°3'38''E) region of Abuja suburbs in the F. C. T., Nigeria. The plant was authenticated and deposited in the National Institute for Pharmaceutical Research and Development (NIPRD), Idu, Abuja where a voucher specimen (No.6738) was given. The collected root was cut into small pieces and air-dried. The dried pieces were pulverized, sieved and stored in a wellventilated area. 1kg of the plant sample was exhaustively extracted using a soxhlet extractor. The marc was extracted successfully using 2.5L each of ethyl acetate, chloroform and chloroform: methanol (ratio 1: 1). The crude extracts were later concentrated to a minimum volume using rotary evaporator (Büchi Labortechnik AG, Switzerland) at 40°C and reduced pressure. They samples were then kept in an airtight container until required for further laboratory analysis.

Phytochemical Screening
Phytochemical screening was carried out on the ethyl acetate, chloroform and chloroform: methanol extracts for the qualitative determination of major constituents using methods previously described [9].

Antimicrobial Screening
Bacterial and fungal isolates were collected from the Department of Medical Microbiology, Ahmadu Bello University Teaching hospital, Zaria, Kaduna State, Nigeria. The bacteria were confirmed using standard biochemical tests according to the Bergey's manual of Bacteriology, while the fungal isolates were identified using fungi chrome test kits [10]. Agar diffusion method was adopted [11].

Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) was determined on the test organisms that were sensitive to the extracts and was done by broth dilution method [12]. Two fold serial dilution of the extract in sterilized broth was made to obtain the concentration of 50.0 µg/ml, 25.0 µg/ml and 12.5 µg/ml. The initial concentration was obtained by dissolving 6.0 mg of the extract in 10mls of sterile broth. Having obtained the different concentration of the extracts in the sterile broth, was observed for turbidity (growth), the lowest concentration of the extract in the broth which shows no turbidity was recorded to the MIC.

Determination of Minimum Bactericidal and Fungicidal Concentrations
Minimum bactericidal concentration (MBC) and Minimum fungal concentration (MFC) were evaluated by plating the bacterial suspensions from individual well at the beginning and at the end of the experiments on Mueller Hinton agar medium for estimation of MBC [12]. The culture from MIC well was taken and streaked on the surface of fresh Mueller Hinton agar in a 90-mm plate with division and incubated at 37°C for 24 hours (bacteria) and 30°C for 1-7 days (fungi) after which the plates of the medium was observed for colony growth, the MBC/MFC were the plates with lowest concentration of the extract without colony growth. Table 1 represents the phytochemical composition of ethanol extracts from the roots of Waltheria indica Linn. Compounds detected included alkaloids, cardiac glycosides, flavonoids, phenolic acids, saponins, steroids, tannins and terpenoids. The action of these compounds working in concert would enhance their defensive properties against a host of pathogenic organisms by way of eliciting antioxidative processes that thwarts pathogen-induced free radicals [29]. The detected compounds were also indicative of their polarity. The mean zones of inhibition of the crude extract against 5 bacterial and 3 fungal species are summarized in Table 2. The ethanol extract of Waltheria indica Linn root were found more active against Candida krusei with 26.0 ± 0.70mm zone of inhibition followed by Corynebacterium ulcerans (25.0 ± 0.35mm) and Streptococcus pneumonia (25.0 ± 0.12mm) respectively, while the lowest value 23.0 ± 1.10 mm was recorded for Candida tropicalis. Ciproflaxin was employed as the drug control standard for bacteria with a zone of inhibition value ranging from 30 to 35 mm, while fluconazole was used as the active antifungal drug control which gave a zone of inhibition ranging from 34 to 35 mm. The mean zones of inhibition of solvent specific root extracts against 8 different microorganisms are depicted in Table 3. The ethyl acetate extract of Waltheria indica Linn root were found to be more active against Using chloroform: methanol, the root extracts were most resistant to Klebsiella pneumonia with a zone of inhibition of 29.0 ± 0.85 mm whereas the least zone of inhibition of 26.0 ± 0.35 mm was observed against Candida tropicalis. The data indicated that the root extract of Waltheria indica Linn was sensitive towards Streptococcus pyrogens and Candida albicans as no zone of inhibition was observed using all three solvent extracts.   Table 4). The MBC and MFC values were roughly the same for the extracts with the exception of 1.0 mg/ml recorded against Streptococcus pneumonia for the ethyl acetate extracts ( Table  5). The MBC and MFC values of the crude extract was 5.0 (mg/ml) across all tested clinical isolates.

Microorganisms
Minimum

Discussion
In this investigation, the discovery of particular secondary metabolites proffers the initial scientific insight into the therapeutic potential of the plant against diseased conditions emanating from a host of pathogens [13,14]. Findings from table 1 suggest that the roots contain a number of secondary metabolites, namely; alkaloids, cardiac glycosides, flavonoids, phenols, saponins, steroids, tannins and terpenoids in all solvent extracts are known individually or collectively for their defensive or signaling functions [15]. Alkaloids are useful neuro-stimulatory agents that also possess analgesic and upper respiratory protective functions amongst others [3,16,17,18]. Steroids enhances the immune system by preventing penetration of many pathogens owing to their antimicrobial properties [13]. The presence of both Alkaloids and Steroids in the root extracts suggests that this part of the plant possesses sufficient activity against disease causing pathogens like S. aureus and S. typhi which are responsible for diarrhea and stomach upsets [19]. Phenolic acids have been reported to exhibit anti-allergic, antiinflammatory, antioxidant, antimicrobial, anticancer and antidiarrheal properties [20,21]. Their presence in the plant extracts suggest a strong and wide range of therapeutic applications whereby they thwart the deleterious effects of disease-causing pathogens by inhibiting their growth and proliferation. The mean zones of inhibition of the crude extract using disc diffusion method against different bacterial and fungal species revealed a high antimicrobial activity against selected pathogenic organisms implicated in several upper respiratory tract infections (Table 2). Notably, against Corynebacterium ulcerans (25 mm) in comparison with the control; Ciprofloxacin (10 µg/ml), the data obtained suggests the possibility for isolating an alternative or new drug candidate from this plant. The antifungal activity was high against Candida tropicalis (24mm) as the plant extracts displayed susceptibility towards Candida albicans and Streptococcus pyrogenes in a manner that suggests a preference towards particular bacteria than fungi. The exhibited antimicrobial spectrum of the crude root extracts is linked to the bouquet of phytochemicals present at varying concentrations. Extensive research revealed that flavonoids possess the inherent ability to deactivate microbial adhesion and cell envelope transport proteins [22,23]. Other specific actions of flavonoid rich plants against pathogenic attack include alterations in microbial fluidity as well as the complete distortion of the pathogens' respiratory and intercalation of the microbial nucleotide sequences, thus preventing microbial replication mechanisms [24,25]. The presence of flavonoids and its synergy with alkaloids alone is highly suggestive of a higher antimicrobial spectrum as alkaloids are reportedly extensively used as pharmaceuticals, psycho-stimulants, narcotics and poisons due to their renowned biologic activities [26]. The activity of alkaloid extracts alone have been investigated in plants like Callistemon citrinus and Vernonia adoensis whereby at concentrations of 1.7mg/ml, it inhibited the growth of Staphylococcus aureus with comparable effects to ampicillin [27]. Other possibilities include the synergies between flavonoids and tannins, both of which have been studied and found to be active against Staphylococcus aureus alongside a host of bacterial pathogens linked with respiratory infections [19,28]. Phytochemicals separated on the basis of solvent extractions (Table 3) revealed that the Ethyl acetate fractions is best suited for treating S. aureus (28mm), K. pneumonia (30mm), C. ulcerans (29mm), C. krusei (31mm) and C. tropicalis (30mm). As depicted in the results shown (table 2), the roots of Waltheria indica Linn is endowed with natural resources to tackle C. ulcerans infections, thus indicating a possibility for treating other infections caused by glutaminerich pathogens like Mycobacterium tuberculosis, thus could be used in treating tuberculosis infections. Tables 4 and 5 data shows the MIC, MBC and MFC of the different extracts of Waltheria indica Linn. The ethyl acetate fraction of the roots had the lowest consistent MIC value (Table 4) against all clinical strains tested than that of any other solvent fraction (0.25mg/ml). The minimum bactericidal and fungicidal concentrations were the same for K. pneumonia and C. krusei (0.5mg/ml for all solvent fractions) and within the 0.5-1.0mg/ml range for all tested fractions and microorganisms (Table 5).

Conclusion
The present study has shown the phytochemical composition and anti-microbial activity of Waltheria indica Linn root. The results authenticates indigenous claims for ethno-medicine in developing countries in addition to improving our depth of knowledge on plant materials useful in the treatment of upper respiratory tract infections. The discoveries in this study provides scientific support for the antimicrobial spectrum from the phytochemical elements within the root extracts of Waltheria indica Linn where the solvent fractions were found to have a significantly high zone of inhibition values when compared to standard drugs ciprofloxacin and fluconazole.