Medicinal Potentials of Virgin Plants: the Case of Beloperone Asclepiadea Nees (Acanthaceae)

Shade–dried Beloperone asclepiadea Nees (whole plant) was extracted by Microwave Assisted Extraction and Cold Maceration using n-hexane, dichloromethane, ethyl acetate and methanol in order of increasing polarity. Phytochemical analysis was run on the extracts followed by antibacterial and antifungal evaluations. Phytochemical screening revealed the presence of glycosides, tannins, flavonoids, saponins, cardiac glycosides, steroids/triterpenoids and alkaloids in the methanol extract. Ethyl acetate and dichloromethane extracts showed the presence of all except flavonoids and saponins respectively while only glycosides, cardiac glycosides and steroids/triterpenes were detected in nhexane extracts. The extracts showed antimicrobial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhi, Bacillus subtillis, Streptococcus pyogenes, Candida albicans and Aspergillus niger with zones of inhibition (ZOI) in the range 30.0±0.0 mm and 11.0±0.0 mm except for dichloromethane extract which showed no activity against S. pyogenes, S. typhi and C. albicans. Inhibition against S. typhi (ethyl acetate extract) and E. coli (methanol and n-hexane extract) have the most competitive ZOI values of 30±0.0 mm compared to 32.0 mm and 40.00 mm respectively for ciprofloxacin at 50 μg/mL. The strongest antifungal ZOI’s are 18.0±0.0 mm against C. albicans (ethyl acetate extract) and 16.5±0.5 mm against A. niger (hexane extract) compared to 30.0 mm at 50 μg/mL by the standard drug, terbinafin. Relatively very low minimum inhibitory concentrations (MIC’s) of 1.5625 mg/mL and 3.125 mg/mL were observed respectively for activity against S. aureus (N-hexane extract) and E. coli (ethyl acetate and methanol extracts) while the lowest antifungal MIC of 12.5 mg/mL was recorded against C. albicans (ethyl acetate extracts). Minimum bactericidal/fungicidal (MBC/MFC) values of 25 mg/mL were observed for S. aureus (n-hexane extract) and E.coli (methanol and ethyl acetate extracts) while 50 mg/mL were recorded for S. typhi, B. subtilis, S. pyogenes and C. albicans by various solvent extracts. These results taken together, confirm medicinal potency of B.asclepiadea against diseases caused by S. aureus, E. coli, S. typhi, B. subtilis , S. pyogenes and C. albicans (in that order) and strongly indicate the presence of bioactive compounds of high therapeutic value.


INTRODUCTION
Since prehistoric times, humans have used natural products, such as plants, animals, microorganisms, and marine organisms, as traditional medicines to alleviate and treat diseases, with the use of plants as medicines traceable to as far back as at least 60,000 years (Shi et al., 2010& Fabricant, 2001. Beyond the positive therapy from ethnic medicines obtained by trial and error people also consumed poisonous plants, which led to vomiting, diarrhea, coma, or other toxic reactions and even death, all experiences being handed down to succeeding generations who become more and more medicinally enriched. Traditional or ethnic medicine (ethno-medicine), also known as, or that metamorphosed into complementary and alternative medicine, still plays key medicinal roles in many countries today (Abdullahi, 2011). It deploys natural products in its crude or semi crude forms, mostly from plants, based on such information inherited from ancestral lineages. It is fast blossoming into orderly regulated systems of medicine in their various forms and despite certain defects, remain a valuable storage area of human medicinal knowledge (Alves & Rosa, 2007;Fabricant, 2001). Ethnic herbal medicines, the major contributors to traditional medicinal practice, have been urbanized over the past thousands of years with respect to methods of preparation, selection of herbs, identification of medicinal materials and the most suitable time for obtaining different plants. Their role in the development of new drugs, affordability, fewer side effects, easy accessibility, natural healing and also dissatisfaction with the results from orthodox pharmaceuticals have kept them in constant use (Bandaranayake, 2006). Herbalists provide drug leads and information on therapeutic potentials of plants which save energy and cost. Today countless active compounds critical to modern 'western' medicine have been extracted from plants, some following their traditional uses, others not. These include drugs such as codeine, morphine, quinine, quinidine, digoxin, vincristrine, vinblastine and atropine among many others. It is estimated that 60% of anti-tumour and anti-infectious drugs already on the market or under clinical trial are of natural origin (Yue-Zhong, 1998). Use of herbal medicines even in developed countries has expanded sharply in the latter half of the twentieth century (Blumenthal et al., 1998 & World Health Organization (WHO), 1999) following their unique chemical diversity which results in diversity in their biological activities and drug-like properties. A single herb or formula may contain many phytochemical constituents such as alkaloids, terpenoids, flavonoids, tannins, glycosides, saponins, steroids etc, and these chemicals may function alone or in combination with one another to produce the desired pharmacological effect (Parasuraman, 2014). The cumulative aftermath of the foregoing is that, by and by, plants increasingly remain the bedrock of human medication and medicinal solutions. Historically, a lot of diseases had no cure until drugs were eventually developed from plants. Up until 1820 malaria still had no cure until quinine was isolated from the bark of the cinchona tree (a plant) and has been used as one of the most effective treatments for malaria to date (Achan et al. 2011). Cancer was the second leading cause of death in the world after cardiovascular diseases. There was no known cure to cancer until the anticancer drug Paclitaxel (Taxol) was derived from the bark of the "pacific yew" tree (still a plant) was discovered in 1962 and was first tested in patients in 1984 (Slichenmyer et al., 1991). It is therefore logical to believe that the cure to today's (and possibly tomorrow's) incurable diseases (like AIDS, Alzheimer's, Arthritis, Cystic fibrosis, Diabetes, Ebola virus, Hepatitis B, Asthma, Parkinson's disease, Poliomyelitis etc) may rest with un-researched plants! Another critical area of concern is the issue of resistance developed by microbes with time to hitherto effective drugs. Penicillin (17) was successfully developed to treat bacterial infections until microbes started resisting it (Spellberg & Gilbert, 2014). Quinine was known to be one of the most effective drugs for malaria, but resistance was reported in the 1980s (Bunnag et al., 1996) and as of 2006, it was no longer used as the best treatment for malaria. Praziquantel is one of the few drugs known to treat schistosomiasis and the parasites are beginning to become resistant (Knobler et al., 2003). Artemisinin another plant based game-changer of an antimalarial has started to show resistance by microbes as of July 2016. Also the last resort for gonorrhea "cephalosporin" has been confirmed to show resistance by microbes in at least 10 countries (WHO, 2018). This brings to fore the need for fresh discoveries and new drug options to tackle microbial resistance. Alternative antimicrobials are urgently needed (Mandal et al., 2009).There are about 250,000-500,000 existing plant species and only a tiny proportion has been scientifically researched for bioactivities (Ngo et al., 2013). Another estimate insists there are about 2.5 million species of higher plants and the majority of these have not yet been examined for their pharmacological activities (Cox et al., 2010). Therefore, there is great potential for fresh discoveries from virgin plants, to provide answers to today's unanswered medical questions. Phytochemicals from these plants could offer huge potential in deriving useful information about chemical structures and new types of action related to new drug development. Virgin plants are plants harvested and used traditionally to treat diseases without their pharmacological effects being studied and without enough information about dosage, effects and risk factors. A lot of virgin plants have been used successfully to treat illnesses. Only a tiny fraction of the existing plant species have been scientifically researched for bioactivities since 1805, others are being used traditionally while the rest are yet to be discovered (Yuan et al., 2016). Many of the medicinal plants whose pharmacological activities provided answers to medicinal questions at various times in the history of disease control, drug resistance and response, from analgesic codeine, antimalarial quinine, antihypertensive reserpine to anticancer taxol, were plants that were at one time considered 'virgin'. Remedy for development of drug resistance by microbial strains and solution to other subsisting medical challenges worldwide will likely reside in the 'bosom' of virgin plants, whose ethno-medicinal potentials may not be known (Pooja & Tannaz, 2017). Virgin plants are most likely the reservoir for antimicrobials against today's incurable diseases and drug resistant strains. Beloperone asclepiadea Nees (Acanthaceae) is a virgin plant that is widespread in the tropical and sub-tropical regions. As far as I know its medicinal potentials have not been investigated despite great medicinal value of plants in the same genera. Phytochemical, antibacterial and antifungal analysis will be done on extracts of the whole plant from four solvents of different polarities. The medicinal potency of the different plant will be assessed by comparing the sensitivity zone of microbial inhibition, of different solvent extracts with those of standard antimicrobial drugs. Further medicinal value will be estimated from the minimum inhibitory concentrations and the minimum bactericidal/fungicidal concentrations of each extract against sensitivity strains. Information from these assays may be the key to providing answers to the contemporary medical question. Positive results will hasten the isolation of implicated bioactive phyto-constituents and may ultimately lead to new drug discovery and formulation.

Collection and identification of plant materials
Beloperone asclepiadea was harvested in July 2019 from the local forests of Ekeh Ayeje area in Edumoga District, Okpokwu Local Government Area of Benue State Nigeria. The plant material was identified by Mr. Namadi Sunusi of the Herbarium of the Department of Biological Sciences, Faculty of Life Sciences, Ahmadu Bello University, Zaria.

Preparation and extraction of plant material
The plant material (2.5kg) was shade-dried and pulverized, after which it was separated into two batches. The first batch was distributed into glass bottles, covered with nhexane, tightly closed and subjected to Microwave-Assisted Extraction (four rounds of three minutes each, after leaving to cool) with the microwave set at defrost. Each jar was then exhaustively 'washed' in sequence with four selective solvents in order of increasing polarity (nhexane, dichloromethane, ethyl acetate and methanol). The combined extracts for each solvent were concentrated and left to dry to constant mass. The second batch was distributed into glass bottles covered with n-hexane and subjected to cold maceration for 72 hours with frequent agitation. The extracted materials were then washed exhaustively with n-hexane. The combined extracts were concentrated and left to dry to constant weight. The same procedure was repeated in sequence using dichloromethane, ethyl acetate and methanol after drying the marc from the preceding solvent.

Culture media and microbes
Antimicrobial screening was done to check the activity against the following microorganisms: (bacteria) Staphylococcus aureus, Escherichia coli, Salmonella typhi, Bacillus subtillis, streptococcus pyogenes; (Fungi) Candida albicans and Aspergillus Niger. Growth media used were prepared according to manufacturer's instructions and include: Nutrient agar (Acoumix TM ), Nutrient broth (HKM), Sabourand Dextrose agar (Titan Biotech), Mueller Hinton agar (Titan Biotech), Sabourand Liquid Medium and Normal saline. They were sourced from the Department of Pharmaceutical Microbiology Ahmadu Bello University, Zaria, Kaduna State, Nigeria. Ciprofloxacin (anti-bacterial) and Terbinafin (anti-fungi) were used respectively as positive antibacterial and antifungal controls.

Sensitivity test for antimicrobial activity by zone of inhibition (ZOI)
The Agar Well Diffusion method (Singleton, 1981, Ghatage et.al, 2014) was adopted. 20ml each of sterile Mueller Hinton Agar prepared according to manufacturer's instruction were poured into sterile Petri dishes and allowed to harden. 2ml of each culture standardized with 0.9% normal salines (McFarland turbidity standard) were swept over each dish and excess discarded. Equidistant wells of about 8mm diameter were bored into the seeded agar using flamed cork borer and labeled. Each well was filled with 100µL of the appropriate extract at 100mg/mL, lower concentrations (50,25,12.5mg/mL) and standard drugs at 50µg/mL. The plates were loaded in duplicate and left for one hour to allow for proper diffusion before incubating upright in the dark. After incubating for 24 hours (at 37 o C) for bacteria and 72 hours (at 30 o C) for fungi they were observed for growth and the diameters of any resulting zones of inhibition formed around wells measured in millimeter scale using a transparent ruler.

Minimum inhibitory concentration (MIC)
The Broth Dilution methods (Cleidson et.al, 2007) were used. The following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625 and 0.1953 mg mL -1 of plant material were prepared by two-fold serial dilution in Muller Hinton agar (for bacteria) and Sabourand Dextrose agar (for fungi) petri dishes. Sterile micro filter paper discs for each of the six organisms were placed on the solidified agar and 10µl of each standardized culture was then added and left for an hour to diffuse. The loaded dishes were incubated in the dark at 37°C for 24 hours (bacteria) and 30°C for 72 hours (for fungi). They were then observed for visible growth. The lowest concentration at which no detectable bacterial or fungal growth occurred was taken as Minimum Inhibitory Concentration (MIC).

Minimum bactericidal/fungicidal concentration (MBC/MFC)
The minimum bactericidal/fungicidal concentration of the extracts was determined from the tubes that showed no visible growth in the MIC determination. The micro filter paper discs bearing the organisms were lifted and seeded on fresh 5ml sterile Nutrient Broth (bacteria) and Sabourand Liquid medium (fungi) in labeled bottles. The incubation cycles were repeated. The lowest concentration of the extracts that showed no visible colony growth (turbidity) on the medium was regarded as minimum bactericidal/fungicidal concentration.

Comparative Solvent Extractive Yields
The results from extraction yields of the four solvents used for the two extraction methods is shown in Table 1. The results is presented in the form of percent extractive yield of each solvent from the two methods

Phytochemical Screening of Extracts
The results of different phytochemical tests carried on the extracts of different solvents for the presence of steroids, triterpenes, tannins, flavonoids, saponins, cardiac glycosides, anthraquinones and alkaloids is presented in Table 2.   Table 3 shows the results of sensitivity tests by zones of inhibition (ZOI) of the four different extracts and positive standards against the selected bacterial strains and fungal strains. ZOI (in millimeter) is presented in mean values from duplicate petri dishes plus or minus Standard Deviation.   Key: + = Growth, -= No growth, -¢ = minimum inhibitory concentration

Minimum Bactericidal/Fungicidal Concentration (MB/MFC) of Extracts
The results for minimum bactericidal/fungicidal concentration are shown below (Table 5) with the lowest concentration that killed organism (MBC/MFC) for the extracts against various microorganisms highlighted.

Comparative Extraction
The results in Table 3.1 shows that cold maceration method, with higher recovery yields, provided better extraction efficiency for Beloperone asclepiadea compared to Microwave Assisted Extraction. Extraction efficiency is dependent on factors such as temperature, surface area of the material, extraction time, solvent polarity, etc. However, almost all the parameters were similar for both methods. Cold maceration is a conventional process done at room temperature whose one advantage over the hot process is its less likelihood to cause degradation of thermolabile compounds present in the plant. It may be suggested that the plant material is laden with thermolabile constituents that may not have been tolerated by the hot MAE process. Sankeshwari et al. (2018) also obtained a higher yield of extracts from CM results from the extraction from the roots of Licorice (Glycyrrhiza glabra) plant. The four solvents have different extractive yields depending mainly on the polarity, with methanol having the highest percentage yield. This is either because there may be plenteous highly polar (as against highly non-polar) phytochemicals in the plant that the earlier solvents could not remove or because methanol can extract both the polar and some of the non-polar compounds. (Dixon & Jeena, 2017). The former reason is supported by the lowest yield recorded by n-hexane in both methods after exhaustive extraction. The greater extractive yield of dichloromethane than ethyl acetate and n-hexane may mean that moderately non-polar constituents are present in higher amounts than moderately polar and strongly non-polar constituents (Barchan et al., 2014 however, consistent with the fact that phyto-constituents in nature usually occur in very low yields far below the demand for bioactive compounds from them used in modern medicine (Ivor, 2000;Beutler, 2009). This is the factor that promotes their artificial synthesis after isolation and characterization. Table 3.2 indicates the presence in B. asclepiadea of cardiac glycosides, saponins, steroids/triterpenes, flavonoids, alkaloids and tannins while anthraquinones were absent. The methanol extract tested positive for all the classes of phytochemicals found in the plant. The ethyl acetate extracts tested negative for anthraquinones and flavonoids while the n-hexane extract only indicated the presence of cardiac glycosides and steroid/triterpenes. The result appears to be in consonance with phytochemical results observed in some plant species of the Acanthaceae family from which active compounds have been isolated (Renjini Haridas et al., 2017;Sultana &Das, 2019 andSathiyabalan et al., 2018). The spread of positive phytochemical results across solvents of different polarities may imply the presence of both polar and non-polar active compounds. However there is more tilting towards highly polar and moderately polar bioactive compounds. A number of vital secondary metabolites with polar groups, especially flavonoids and poly phenolic compounds with their high hydroxyl content may be involved. They are precursors for drug synthesis due to their peculiar activities as good scavengers of free radicals group in them (Kumar and Pandey, 2013). The same goes with saponins which are known to possess both antimicrobial and anti-inflammatory activities. The positive test for alkaloids and steroids/triterpenoids shows likely concentrations of these classes of phytochemicals known for their physiological, pharmacological and hormonal activities (Lu et.al, 2012;Einbond, 2009). B. plumbaginifolia, a related plant, had some isolated compounds with reported antioxidant, antiinflammatory and antinociceptive properties (Rajasekaran et al., 2012).

Antimicrobial Activity
From Table 3.3 almost all the extracts showed some antibacterial activity at a concentration of 100mg/mL except that the dichloromethane extracts showed no antifungal activity against Candida albicans and no antibacterial activity against Streptococcus pyogenes and Salmonella typhi. It is also the only extract that showed no activity against any of the microorganisms at a concentration of 12.5 mg/mL. The other extracts had very interesting antimicrobial ZOI's (at 100 mg/mL and at least up to 12.   asclepiadea having showed high activity against some of the microbes implicated in those unanswered medical questions may provide a lead to finding a new way around these subsisting medicinal problems. The plant obviously has to be investigated further not only for activity against more bacteria and fungi but more importantly for isolation and characterization of bioactive compounds.

CONCLUSIONS AND RECOMMENDATIONS Cold Maceration
Extraction of B. Asclepiadea proved to be the better method for the extraction of the plant than microwave assisted extraction, suggesting that the plant material is laden with thermolabile constituents that may not have been tolerated by the hot MAE process. The comparatively high extractive yield of methanol and dichloromethane compared to n-hexane and ethyl acetate may mean preponderance of highly polar and moderately non polar phyto-constituents as against non-polar and moderately nonpolar ones. This is further confirmed by the positive phytochemical results for cardiac glycosides, saponins, steroids/triterpenes, flavonoids, alkaloids and tannins and their spread across solvents of polarities that favour highly polar and moderately polar bioactive compounds. The antimicrobial results showed strong activity against both gram positive and gram negative bacteria S. typhi, E. coli, B.subtillis, S. aureus, S. pyogenes and fungus C. albicans with zones of inhibition with competitive values to those of the standard drugs. The relatively very low MIC's of 1.5625 mg/mL observed for activity against S. aureus, 3.125 mg/mL for activity against E.coli and 6.25 mg/mL for activity against S.typhi, B.subtillis and S.pyrogenes almost conclusively prove potency against corresponding bacterial infections as (S. aureus) meningitis, sepsis, osteomyelitis, (E. coli) urinary tract infection, cholagitis, choleeystitis, (S. typhi),typhoid fever, (B. subtilis) food poisoning, meningitis, (S. pyogenes) tonsillitis, scarlet fever and pharyngitis. There is also great optimism against candidiasis, skin infections, urinary and genital yeast infections with the lowest antifungal MIC of 12.5 mg/mL recorded against Candida albicans.
Medicinal potency for the plant may stretch beyond antibacterial and antifungal to cover anti-inflammatory, antioxidant and other activities. Related plants have been reported both to possess antioxidant and anti-inflammatory activities (Sathiyabalan et al., 2018;Shafique & Qureshi, 2015) and to show high activity against selected microbes that appear to follow a trend (Sharma & Kumar, 2016;Asifjavaid et al., 2014). Finally high potency of the plant extracts against S. aureus, E, coli, S. typhi, B. subtilis , S. pyogenes and C. albicans raises hope of a fresh inroads around drug resistance to diseases linked to the microbes, and to others with no known cure. The plant is currently being further investigated for activity against more microbes and for isolation and characterization of implicated bioactive compounds. Recommended areas of further research include invivo pharmacological activity tests and medicinal risk assessment.