Dynamics to Enhance the Conservation of Locally Threatened Medicinal Plant Species (Maerua Oblongifolia) Through Phytochemical Analysis and Antibacterial Study

Introduction: For generations, many plants were extracted to nd drugs and to discover novel and new chemical structures from extracts. Maerua oblongifolia (Forsk.) A. Rich, “Sangganaa” in Dawuro language is a locally threatened traditional medicinal plant, which is traditionally used to treat diarrhea. The present study aimed to check the chemical contents of the different plant parts for possible shifting of the extensive use of the root to other parts of the plant. Methods: Phytochemical screening tests were carried out by standard procedures to investigate various classes of secondary metabolites. Roots, root bark, stem bark, and leaves were extracted by maceration in aqueous and ethanol to get crude extracts. Antibacterial effect of extracts against Escherichia coli and Salmonella thypi was evaluated by the disc diffusion method. Results: The phytochemical analysis showed the presence of alkaloids, saponins, tannins, phenols, carbohydrates, and proteins in the tested parts of the plant. Antibacterial activity displayed the extracts of M. oblongifolia against tested bacteria strains in dose dependent manner. Among tested extracts, roots and stem barks ethanol extracts showed potential antibacterial activity, with zone of inhibitions (19.0 ± 1.15, 17.0 ±1.00) in 100 mg/mL, and (17.3 ± 0.57, 15.0 ± 1.00) in 50 mg/mL, respectively. Conclusion: The nding showed a possibility to shift stem bark instead of roots in the use of traditional medicinal practices to ensure conservation and sustainable use of Maerua oblongifolia. The nding also supports the traditional use of the plant’s root or stem bark for the treatment of diarrhea.


Introduction
For generations, many plants were investigated to nd new drugs and combat drug-resistant microbials and the appearance of undesirable side effects of antibiotics. In this way, the search for new antimicrobial agents and the discovery of novel chemical structures from plant extracts has continued. Phytochemical screening of traditional medicinal plants is very important in identifying new sources of new natural products for drugs (Savithramma et al., 2011). Around 80% of the plant products discovered to date are said to be of plant origin and their the annual market for herbal products had sales exceeded the US $65 billion (Savithramma et  Maerua oblongifolia (Forsk.) A. Rich (Capparaceae) is a woody shrub/tree plant. Its traditional use of root part was known in the study area for generations (Agize et al., 2013). This part was pounded; chewed; administered as dry bath, and taken either with other plants or alone in the form of drink; smoke (except for pregnant women) orally, nasally or anally. Traditionally, they use this plant's root to treat anthrax, stomachache, severe abdominal cramp, hook worm, body swelling, mump, tetanus, eye disease, liver cirrhosis, gonorrhea, meningitis and for different disease of both humans and cattle (Agize et al., 2013;Ekero et al., 2018). Most of the traditional medicinal use practice of the area has similarity with other areas. It was approved that the plant is used to cure various diseases such as fever, stomach ache, skin infections, urinary calculi, diabetes mellitus, epilepsy, pruritis, rigidity in lower limbs, and abdominal colic  (Meragiaw, 2016) and act as alterative and energy stimulant (Savithramma, 2016).
It is indicated by local informants and herbalists that the species has been endangered in the area because of the widespread use of its root as a detergent, medicine, and for other purposes. Its growth in water stress areas, stony places of lowlands 300m-1350 m above sea level (Kuria et al., 2017), unsustainable harvesting (Agize et al., 2013), and not domesticated being found only in the wild were also some of its threatened conditions. In general, the importance/relevance of this research work is justi ed since the species is locally threatened, the multiple uses that target material derived from the root part, harmful harvesting methods, and the variety of health problems traditionally treated by the remedy derived from it.
Thus, the activity was done to see whether the possibility to shift root use to other parts (root bark, stem bark and leaf) of the plant is plausible and worth considerations in future conservation efforts. In other word, the research attempted to check why knowledgeable persons focus on root rather than other parts i.e, to check the indigenous knowledge of using root because of the secondary metabolite found concentrated in the root or/and the continued use of this part because of acquisition from parents and others.
The study aimed at identifying the best strategy for conservation and sustainable utilization of the locally threatened traditional medicinal plant, M. oblongifolia, through an ethnobotanical knowledge-led phytochemical analysis and antimicrobial testing using extracts from its different parts.

Preliminary Ethnobotanical Study
The ethnobotanical information survey was conducted by using pre-prepared semi-structured individual interviews from September 2019 to April 2020. The sample size, 385 was determined following Cochran (1963) formula and snowball technique was used to nd traditional medicinal plants practitioners for interviews. The semi-structured interview questions comprise traditional medicinal plants ailments/ disease treated, how they use, the habit, part and source of the plant from where they collect, and its conservation status following fore scholars (Martin, 1995;Cotton, 1996). Ethnobotanical knowledge on high delity level medicinal plants whose roots are known to have medicinal use value, collected from wild and locally threatened, was selected. One of these plants M. oblingifolia was informed that it is used to treat both human and domestic animals.

Plant Material Collection
Fresh leaf, root, root bark, and stem bark of M. oblongifolia were collected from its natural habitat of Dawuro, Southwest Ethiopia, from August 2020 to December 2020. Botanical identi cation of the plant was carried out using Ethiopia's ora and compared with voucher specimens at the National Herbarium of Ethiopia and authenticated by experts there. Finally, the identi ed triplet sample voucher specimen with voucher number MA0306/2020 was deposited there at the National Herbarium of Ethiopia at Addis Ababa University, Herbarium of Arba Minch University, and Mini-Herbarium of Dawuro Historical and Cultural Museum.

Preparation of Plant Extracts
The collected leaves, roots, root barks, and stem barks of M. oblongifolia were transported to Organic Chemistry Laboratory, Wolaita Sodo University, Ethiopia, and washed with tap water to remove unnecessary particles and external moisture wiped out with a dry cloth, and dried under shade at a room temperature. Brie y, the air dried plant materials were then carefully chopped, crushed, and pulverized into smaller fragments in mortar and pestle and nally ground into ne powder by using an electrical grinding mill.
Grounded powder of root, root bark, stem bark, and leaves of 150g was extracted with distilled water (aqueous) and 99.8% ethanol by maceration separately, while shacking by electronic shaker at speed 230 rpm at room temperature for 24 hours. The solutions were ltered by suction ltration. The aqueous ltrates were suspended in the water bath at 60 0 C at room temperature to concentrate a crude extract.
The ethanol ltrates were concentrated by a vacuum rotary evaporator at 60 0 C. Finally, the crude extracts were collected by labeled sterilized containers and kept under a deep freezer until further experiment (phytochemical analysis and antimicrobial activities testes).
Phytochemical analysis of M. oblongifolia crude extracts Preliminary qualitative phytochemical analysis was carried out to detect the presence or absence of secondary metabolites in ethanol and aqueous leaf, stem bark, root bark, and root extracts. These extracts from distilled water is blue back and yellowish/orange colour from ethanol solvent systems. The qualitative test was carried out in each case.

Test for Saponins
About one gram of ethanolic and 1 g of distilled water crude extract was boiled separately with 5 ml of distilled water, and then it was ltered. To the ltrate, about 3 ml of distilled water was further added and shaken vigorously for about 5 minutes. Frothing, which persisted on warming in each case, was taken as evidence for the presence of saponins (Gul et

Test for Phenolic Compounds
The crude ethanolic and distilled water extract (20 mg) was dissolved separately in 5 ml of distilled water, and few drops of neutral 5% ferric chloride solution were added. A dark green color in each case was

Test for Flavonoids
About 0.2g of ethanolic crude extract was dissolved in water and ltered; to the ltrate, 2 ml of the 10% aqueous sodium hydroxide was later added to produce a yellow coloration. A change in color from yellow to colorless on the addition of dilute hydrochloric acid was taken as evidence for the presence of avonoids (Gul et  The Makwar standard solution formula M 1 V 1 = M 2 V 2 , was used to determine the concentration of extracts and solvents. The four parts of ethanol and water extracts, total of eight stocks was prepared. The rst 100% was used as stock; 100%=15 ml; 50%=20 ml; 25%=30 ml and 12.5%=40 ml were used in each case. The preparation of a petridish containing 15-25 mL agar, bacteria (E. coli and S. typhi) at a known concentration is then spread across the agar surface and allowed to establish. A 6 mm paper disk containing a known volume of the test substance is then placed in the center of the agar and the dish incubated for 24 h. At this time the "cleared" zone (zone of inhibition) surrounding the disk was measured and compared with zones for standard antibiotics (Bacitracin 10 PK07 sensitivity disc). On the other hands, data from this assay is typically presented as mean size of zone of inhibition.

Result Preliminary Ethnobotanical Information
Most of traditional medicinal practitioners realized that M. oblingifolia is one of the plants extensively used for traditional medicine. It was informed that its root is used to treat more than 13 diseases like diarrhea, stomachache, severe abdominal cramp, body swelling, mamp, tetanus, meningitis, eye disease, liver sirrhosis, gonorrhea, hook worm, and anthrax and for different disease of both human and cattle.
They extract its constituents using water and "Harak'iya" (local alcohol). They also provided information that the plant was threatened because of its roots were targeted for different purposes including the extensive use as traditional medicine and detergent (local soap) by the community.

Preliminary phytochemical screening of M. oblongifolia extract
The biologically active crude extracts of leaves, roots, root barks and stem barks using the powder of the medicinal plant, M. oblongifolia, which had high delity level was exhaustively extracted with distilled water and ethanol at room temperature ( Table 1). The yield also varied in color. The aqueous was suspended in the air of water bath (HWS-24volt220v/50HZ/Watts 1000w) 60 0 C at room temperature to give (13.1 g, 8.73%) Blueblack/brownish crude extract. The ethanol extract solution was ltered and concentrated by a vacuum rotary evaporator at 60 0 C yielding Orange/yellowish crude extract (16 g, 10.67 %).

Antibacterial Activity
Testing methods in detecting antimicrobial resistance record of the plant extract resulted for the two bacteria using control of the antibiotics (antimicrobial agent) discs to see the range of resistance (Table 3).

Antibacterial Activity
The antibacterial activity of the phytochemical compounds in the root, root bark, stem bark and leaf ethanol and aqueous solvents extracts of M. oblongifolia were checked using Bacitracin as a standard reference drug on E. coli and S. typhi. Testing methods in detecting antimicrobial resistance record of the plant extract resulted for the two bacteria using control of the antibiotics (antimicrobial agent) discs to see the range of resistance (Table 3) Phytochemical compounds are determined as secondary metabolites that pose various properties including antimicrobials, antioxidants, antiviral and many more (Muhongo et al., 2021). The extracts derived from ethanol were more active than aqueous extracts. This may indicate that the non-polar active principles are responsible for the antimicrobial activity in M. oblongifolia (Bhalakiya and Nainesh, 2019). Alkaloids have been extensively researched because of their biological activity and medicinal uses (Thawabteh et al., 2019). The higher level of alkaloids indicates its signi cance and application in treatment of a broad range of infectious diseases (Muhongo et al., 2021). Therefore, though isolation and characterization are yet not done, from qualitative testing and from literature sources, it is projected that the alkaloids in this plant might be bioactive compounds that may result in its effectiveness shown by inhibition of the microbes tested.
The phytocompounds present in Maerua oblongifolia are a potential source of chemotherapeutic compounds. In this study, the root and stem bark were found to contain the most phytocompounds validating their traditional use in the treatment of various ailments such as diarrhea and other bacterial infections. The ndings over and above supporting the traditional use of the plant for the treatment of diarrhea, favored the hypothesis that shifting from the traditional use of the root to that of the stem bark through sustainable harvesting to ensure conservation and sustainable use of the species. This study can also be applied to other traditional medicinal plants in which the species are threatened due to unsustainable use of more sensitive plant parts by shifting to less sensitive parts after checking the effectiveness. Further research is, however, required to optimize the biomedical applications of the plant. The two solvents used in this study were able to extract different bioactive compounds and this constitutes vital information for those wishing to extract compounds from the plant for further research as their presence was observed in all the plant parts to isolate, identify, characterize, and elucidate the structure of these bioactive compounds. The use of different solvents may also be considered as solvents are selective for the extraction of speci c compounds.
The results from this study demonstrate that M. oblingfolia root and stem barks have abundant secondary metabolites which are largely utilized in traditional medicine to combat and cure numerous infections including diarrhea. Qualitative and quantitative analysis indicates that the M. oblingfolia root and stem barks are potential sources of phytochemicals that could be used in drug discovery and development. Ethanol solvent extraction yields maximum phytochemical compared to the other solvents and therefore recommended for the extraction of plant material from this species.
The antibacterial testing of the M. oblingfolia root and stem barks extracts ensured the inhibition potential of diarrhea-causing bacteria. However, further con rmatory studies followed by isolation and characterization of the active antimicrobial compound (s) of the plant that are responsible for the observed microbial suppression is needed before it is recommended for diarrhea drug search and discovery. Since, both parts do have potential of inhibition; it has possibility of using stem barks instead of root of the plant to avoid threatening condition of traditional harvest.

Conclusion
Maerua oblongifolia is being overexploited and is in danger of becoming extinct in local level due to root harvesting for different purposes. In this case, since the bioactive extracts from the root and stem barks of the plant were tested and con rmed based on the ethnobotanical leads, extraction and application to treat diarrhea, this information will have practical application in conserving the species and facilitating continued sustainable utilization. That is since most of the bioactive compounds present in the root are also found in the stem bark in su cient quantities, it is possible to harvest ascertain sector of the stem bark while leaving the other sector and other parts, especially the underground part (root and root barks) of the plant intact to regenerate itself.
Finally, vitality works that can save those threatened plants with medicinal use through assessing indigenous knowledge about the use and status of medicinal plants before the knowledgeable people leave or die combining it with ethnobotanical knowledge-led composition analysis and antibacterial testing is a better strategy to conserve threatened medicinal plants. This must be followed by awareness raising mainly targeted to traditional medicinal plants practitioners to shift the plant part they use those known to be less destructive and suited to sustainable harvesting thereby ensuring continued use and conservation of the target plant species.

Declarations
Ethics approval and consent to participate "Not applicable".
Consent for publication "Not applicable".
Availability of data and materials "Not applicable.
Competing interests "The authors declare that they have no competing interests".

Authors' contributions
Mathewos Agize Ante conceived and designed the study; collected specimen, performed the experiments; analyzed the data and written the draft of manuscript. Zemede Asfaw WoldeMariam, Sileshi Nemomissa Duguma, and Tizazu Gebre Alemayehu helped to design the study and supervised the study, and edited the manuscript. Mathewos Anza Alemu helped to perform phytochemical analysis study and edited the manuscript. Tizita Lema Bargene and Mintiwab Abrham Dana helped to perform antibacterial testing study. All authors read and approved the nal manuscript.