Study Design: Experimental study design
Study Area
The study was conducted in two rural districts of Sironko and Bulambuli districts in the Elgon sub-region of eastern Uganda and are thought to have a range of plant species[15]. The distances between Sironko and Bulambuli districts and Mbale city and Kampala, the capital of Uganda are 24.7 km and 55.4 km, and 275.9 km and 306.8 km, respectively[23,24]. Sironko district is bordered to the north by the Bulambuli district, the northeast by the Kapchorwa and Kween districts, the east by Kenya, the southeast by the Bududa district, and the southwest by the Mbale and Bukedea districts. To the north, east, and west of Bulambuli district are Nakapiripirit, Kapchorwa, and Bukedea districts respectively. (Figure 1). The average elevation of these districts is 3996 feet (1,218 meters)[23,24]. According to [18], the average annual temperature is 24.4 o C, while there is between 920 and 1650 millimeters of rain per year on average [18]. The study was conducted in seven villages located at latitudes and longitudes indicated for each as Zema (1.212oE, 34.31oN), Suguta (1.24oN, 34.37oE), and Jewa (1.284oN, 34.31oE) in the Bulambuli district, Nakidoba (1.26o N, 34.49o E), Bulwala (1.25o N, 34.43o E), Miwu (1.21oN, 34.34o E), and Madaya (1.21oN, 34.37o E) in Sironko districts. The majority of the custodians of traditional herbal knowledge were elderly, ranging in age from 55 to 75, and they claimed to have been residents of this region for their entire lives. They have accumulated a broad range of ecological knowledge about the different medicinal plants, as shown by this, over time. However, because they are older than the recommended age range (31–45 years), this puts the sector's sustainability in danger, and grave concerns about collapse continue to be raised[13,21]. As a result, an ethnobotanical study was carried out between September and November 2022 in seven villages, including Zema, Suguta, and Jewa in the Bulambuli district and Nakidoba, Bulwala, Miwu, and Madaya. (Figure 1). The selection of these study sites was shaped under the guidance of local leaders and elders, who, to the best of their knowledge, believed they had the most experienced herbalists.
Climate
The Mount Elgon sub-region primarily experiences a tropical humidity-type climate. A seasonal oscillation of dry northwesterly and moist southwesterly air streams is defined[21]. About 23 °C is the average yearly temperature. Additionally, the average minimum and maximum temperatures are 15 °C. The rainy season starts around March and ends in December[22]. The weak bimodal pattern of mean annual precipitation in the Elgon sub-region is that January through March are the hottest months of the year, while July and August are the coolest. The annual average precipitation is about 1500 mm. In the Mount Elgon sub-region, there is a faint bimodal pattern in the precipitation Orographic factors and attitude are the main causes of rainfall variations[21].
Soils
The soil structure of Mount Elgon is typically deep and composed of volcanic ash as a result of a single weathering cycle[23]. One significant characteristic stated is that the carbonatite dome's structure makes the soils in this area very variable[24]. The soil types of Mount Elgon have three major sources, according to research[25]. In the beginning, the presence of volcanic ash and agglomerates beneath volcanic ranges, hills, and pediments contributed to the formation of the soils. Rocks that have undergone metamorphism serve as representations of the degraded Gondwanan surface, where some of the soil comes from. Thirdly, another percentage of soil comes from mixed volcanic-metamorphic rocks
Demographic characteristics
The Bagisu, also known as the Bamasaba, make up the majority of the population in this sub-region and are the seventh-largest ethnic group in Uganda [26]. The majority of the locals are peasants who cultivate a variety of multi-season vegetable crops as well as crops like maize, coffee, potatoes, banana (matoke). The region is also reported for having subpar healthcare facilities, primarily Health Centers III and II, as well as a general dearth of institutions specifically designed to treat cancer cases. This makes therapy less accessible, expensive, and unaffordable for a culture that is predominately rural due to its distance from the Uganda Cancer Institute. Therefore, majority of the communities rely and utilize herbal medicine to meet their healthcare needs since they have trouble getting access to standard prostate cancer treatments and because most of their places are isolated and hard to reach areas especially during rainy seasons. Observations of prostate cancer patients who rely on Rhoicissus tridentata revealed a significant reduction in PSA with respect to other plants and the need to avail of the phytochemical constituents responsible for these anticancer potentials. This sparked curiosity on understanding plant phytochemical composition in these medicinal plants used in the region in the management of prostate cancer.
Experiment design
Collection of Plant Materials
Plant materials for the experimental investigation were collected from the slopes of the Egon sub-region in January and February 2023, notably from the Sironko and Bulambuli districts from a total of seven villages (Figure 1).
Processing of the Plant Material
The plant materials were cleaned, cut into small pieces, and air-dried until constant weight was obtained in the Biology Laboratory at the Islamic University in Uganda. Then, they underwent qualitative and quantitative examinations at the Natural Chemotherapeutic Laboratory and the Chemistry Department of Makerere University, respectively. The materials were dried and then processed into a coarse powder using a wood powder blender (Model BRONICA).
Extraction Process
The serial extraction approach, with already been established methods by [22,23 with modifications, were adopted. The coarse powder was first soaked in methanol and water as solvents in a series of extraction techniques. The plant rhizomes were gathered, cleaned, let air dry, and then ground into a coarse powder. To enable the extraction of active chemicals, exactly 150g of the powdered materials were weighed using a digital scale (Model I1C28 RAGWAG) and then steeped in 1500 mL of diethyl ether (petroleum ether) in an Ehlmeyer flask for 72 hour [29]. Every two hours during the procedure, the mixture was shaken to facilitate the extraction process during the day. It was concentrated at 40 oC and left there to cool until the following day. Using a freezer dryer, the aqueous extract was concentrated. Both the methanol and aqueous extract yields were calculated. For the duration of the study, all of the samples were maintained in the refrigerator after being sealed in airtight containers.
Determination of the Phytochemical Composition
Qualitative Tests
These tests were conducted following the procedure laid down by Kuruppu et al [31]and Farooq et al[33], with modifications. Some of the chemicals that were tested were phenolics, flavonoids, coumarins, terpenoids, tannins, saponins, phlobatannins, anthraquinone, anthocyanins, steroids and sterols, alkaloids, glycosides, cardiac glycosides, phytosterols, cholesterols, proteins and amino acids, carbohydrates, terpenoids, triterpenoids, diterpenoids, fixed oils and fats, volatile oils, and carotenoids. Test procedure
Test for Phenol: Ferric chloride test: To 2 mL of plant extract, a few drops of a 5% ferric chloride solution were added, and the appearance of a dark green or blue-black color indicated the presence of phenol.
Test for Tannins: Braymer’s test: 1 mL of the extract was mixed with 3 mL of water and heated in a water bath. The mixture was filtered, and a 10% ferric chloride solution was added to the filtrate. A dark-green color indicates the presence of tannins.
Test for Phlobatannins: HCl test: 2 mL of extract was added to 2 mL of 1% HCl and boiled. The appearance of a red precipitate shows the presence of phlobtannins.
Test for alkaloids: iodine test: 3 mL of the extracts were dissolved individually in dilute hydrochloric acid and filtered. To the filtrate, 3 drops of iodine were added. The appearance of a blue color that disappears on boiling and reappears on cooling shows the presence of alkaloids.
Test of saponins: Foam test: 0.5 g of extract was shaken with 2 mL of distilled water. Formation of frothing (appearance of creamy misses of small bubbles showed presence of saponins)
Test for flavonoids: alkaline reagent test 1 mL of extract was added to 2 mL of a 2% sodium hydroxide solution. An intense yellow color that becomes colorless with the addition of dilute acid signifies the presence of flavonoids.
Tests for anthraquinone: Bontrager’s test: 10 mL of 10% ammonia solution was added to a few of the filtrates (shake vigorously for 30 seconds). A pink, violet, or red-colored solution indicates that anthraquinone is present.
Test for anthocyanins: HCl test: To 2 mL of extract, 2 M HCl was added (plus a few drops of ammonia). The appearance of the pink-red solution turning blue-violet after the addition of ammonia showed that anthocyanins are present.
Test for terpenoids: 0.5g of the extracts was added to 2 mL of chloroform and evaporated to dryness. 3 mL of concentrated sulfuric acid was added. The appearance of a reddish-brown color in the interphase will indicate the presence of terpenoids.
Test for Triterpenoids: Salkowski test: To the 2 mL of extract were added a few drops of concentrated sulfuric acid (shaken well and allowed to stand). The appearance of a golden yellow layer (at the bottom) signifies the presence of triterpenoid.
Test for diterpenes: Copper acetate test: To 2 mL of extract, 4 drops of copper acetate solution were added. The appearance of the emerald green color shows that diterpenoids are present.
Mix 5 mg of extract with 2 mL of chloroform, and then add the same amount of concentrated sulfuric acid along the sides of the test tube. This is how you test for steroids and sterols. Once the upper layer turns red and the lower layer turns yellow with green fluorescence, it indicates the presence of the steroids and sterol compounds in the extract.
Test for Quinones: HCl test: 1 mL of extract was added to a few drops of concentrated HCl. The appearance of the green color shows that quinones are present.
Test for fixed oils and fats: Spot test or stain test: A few little quantities between the filter papers. The observation of oil stains on the paper shows that fixed oils are present.
Test for volatile oils: Fluorescence test: The extract was applied to the filter paper. A transparent appearance (oils and resins) showed that volatile oils are present.
Test for Glycosides: Bontrager’s Test 2 mL of hydrolysate was added to 3 mL of chloroform, shake well until the chloroformLayer separated, and then add 10% ammonia. The appearance of a pink-colored solution indicates the presence of glycosides.
Test for Cardiac Glycosides: Keller-Killani test: 1 mL of extract, 1.5 mL of glacial acetic acid, 1 drop of ferric chloride solutions, and finally concentrated sulfuric acid (alongside the test tube). A blue-colored solution in the acetic acid layer signifies that cardiac glycosides are present.
Test for Coumarins: Sodium hydroxide test: 2 mL of the plant extract was added to 10% NaOH and then chloroform. The appearance of the yellow color shows that coumarins are present.
Test for proteins and amino acids: Millon’s test: 2 mL of the extract was added to a few drops of Millon’s reagents. The formation of white precipitates reveals the presence of proteins and amino acids.
PriceTest for Carotenoid: Carr-price Reaction: 10 mL of extract was evaporated to dryness, and a few drops of a saturated solution of antimony trichloride in chloroform were added. A blue-green color eventually turned red, showing that carotenoids are present.
Test for carbohydrates: starch test: 1 mL of extract was dissolved in 5 mL of distilled water and filtered. The appearance of acne coloration showed that starch was present.
Test for reducing sugar: Benedict test: 0.5 mL extracts were dissolved individually in 5 mL of distilled water and filtered. Then 1 mL of Benedict solution was added and boiled for 1 minute. The appearance of a green, yellow, or red color signifies that reducing sugars are present.
Test for phytosterols: Salkowski test: 1 mL of extract was added to a few drops of concentrated sulfuric acid (shake well and allow to stand). The red color in the lower layer signifies the presence of phytosterol.
Test for cholesterol: acetic anhydride test: 0.5 mL of extract, 2 mL of acetic anhydride, and then 2 mL of concentrated sulfuric acid. A change of color from violet to blue or green shows that cholesterol is present.
Test for resin: Acetic anhydride test: 0.5 mL of extract was added to 2 mL of acetic anhydride and then 1 mL of sulfuric acid. The change from orange to yellow indicates that resins are present.
Test for carboxylic acid: effervescence test 1 mL of extract was added to a few drops of sodium carbonate powder. The appearance of effervescence signifies the presence of carboxylic acid.
3.5.3.1 Quantitative Phytochemical Determination
The determination of phenols, tannins, alkaloids, flavonoids, saponins, carotenoids, glycosides, saponins, and terpenoids was based on ultraviolet spectrometry (UV spectrophotometry) as per the protocol earlier laid down by [34][35][36][36][37] [38] [39], with modifications.
Determination of total Polyphenols
based The total polyphenol was determined based on the guidelines earlier laid down by [38] and [39], with modifications. The total phenolic compounds were calculated using the Folin-Ciocalteu reagent. In a nutshell, test tubes containing 100 mg of samples were filled with 10 mL of 50% methanol and divided into two portions of 5 mL each. To ensure thorough mixing, the samples were physically shaken for 30 to 40 minutes. The sample extracts were centrifuged for 10 minutes at 300 rpm and then kept at -20 o C The extracts were decanted into test tubes, and 5 mL of distilled water was used to dilute 5.0 mL of the Folin-Ciocalteu reagent (2N). 0.1 mL of distilled water, 0.25 mL of diluted Folin-Ciocalteu reagent, and 1.25 mL of 20% sodium carbonate were used to dilute each sample in three equal parts. After the setups have stood for 35 minutes, the tubes are vortexed for another 15 minutes. The absorbance was measured at a wavelength of 725 nm. In order to create a standard solution, the following concentrations of garlic acids were made: 10, 50, 100, 250, 300, 350, 400, 450, and 500 mg/mL in methanol. The amount of phenol in the sample served as the basis for the creation of calibration curves. Gallic acid equivalents (mg GAE/g dry fruit weight) were used to translate the results into mg of dried extract.
Determination of total Tannins
Each powdered sample was weighed out to a total of five hundred milligrams (500 mg), and then it was placed into a 100-mL plastic bottle. In a mechanical shaker, 50 mL of distilled water was added, and it was agitated for an hour. After filtering, the mixture was produced to the proper consistency in a 50-mL conical flask. The filtrate was then pipetted out in 5 mL portions into a tube, where it was combined with 3 mL of 0.1 M FeCl3 in 0.1 N hydrochloric acid and 0.008 M potassium ferrocyanide. Within 10 minutes, the absorbance at 530 nm was measured with a spectrophotometer[38]. The stock solution was prepared by dissolving 100mg of tannic acid (Sigma) in 1 L of distilled water in a calibrated flask. Standard solutions were obtained by appropriate dilution of the stock solution with distilled water. 1.485g of 1910-phenanthroline monohydrate (Sigma) in distilled water and diluting 500 mL with the same. From this stock solution, a standard solution of 0, 1.5, 0.3, 0.6, 1.2, 1.5, 1.8, 2.1, and 2.4 mg/mL was prepared. The concentration curves were generated at 530 nm, and from these solutions, the concentration of tannins was determined.
Determination of total Flavonoids
A technique described by[39] was used to spectrophotometrically determine the total flavonoid. Test tubes were filled with 2.0 mL of the ethanol extract and received an equal amount (2 mL) of a 2% AlCl3 solution. After one hour of incubation at room temperature, the solution's absorbance at 420 nm was measured. Rutin was dissolved in 100 mL of methanol as a stock solution, or at a concentration of 1 mg/mL, to create a standard curve. With distilled water, the stock solution was diluted to concentrations of 0, 1.5, 0.3, 0.6, 1.2, 1.5, 1.8, 2.1, and 2.4 mg/mL. Rutin equivalents (mg rutin equivalents per g) were then used to express the results. After that, the total flavonoid contents were determined. Rutin equivalents (mg rutin equivalents per g) were then used to express the results. Using the calibration curves that were produced, the following equation was used to determine the total flavonoid levels as rutin (mg/g).
Determination of total Alkaloids:
The protocol outlined in the preceding sentence by[40] was modified for the determination of alkaloid. A finely powdered portion of the plant material (100 mg) was then extracted continuously for 24 hours with methanol (Soxhlet). After filtering the extraction, methanol was vacuum-vaporized in a rotating evaporator at 45 oC until it was completely dry. Some of this residue was filtered after being partially dissolved in 2N HCl. It was then transferred to a separatory funnel; 1 mL of this solution was three times rinsed with 10 mL of chloroform. The pH was changed by adding 0.1 N NaOH to the mixture. The solution was supplemented with 5 mL of phosphate buffer and 5 mL of BCG. 5 mL of phosphate buffer and 5 mL of BCG were added to the solution. Shaking the mixture vigorously allowed the complex to form, which was then extracted using 1, 2, 3, and 4 mL of chloroform. The extract was gathered in a 10-mL flask and diluted with chloroform until volume was reached. At 470 nm, the complex's absorbance was measured. Atropine standard solution was properly measured into liquots (0.4, 0.6, and 1.2 mL), and each was then transferred to separator funnels to create the stock solution. Bromocresol Green (BCG) solution and pH phosphate buffer were also added in amounts of 5 mL each, and the mixture was shaken with 1, 2, 3, and 4 chloroforms. At 417 nm, the absorbance was measured, and using calibration curves, the concentration of alkaloids was calculated.
Determination of total Terpenoid
The procedure previously described by[41] was modified to determine the total terpenoid content (TTC) of the extract of Rhoicissus tridentata (L..f.) Wild and R.B. Drumm. 100 mg of pulverized plant material were then continuously extracted with methanol for 24 hours (Soxhlet). The extraction was filtered, and methanol was dried by vacuum-sealed rotary evaporation at a temperature of 45 o C. 1 mL of the extracts were diluted with 2 mL of chloroform. After fully vertexing the sample mixture, it was left for three minutes. The mixture was then treated with 200 l of concentrated sulfuric acid (H2SO4) and incubated for 1.5–2 hours at room temperature in the dark. During incubation, a reddish-brown precipitate was created in the mixture. The supernatant was then carefully decanted without disturbing the precipitate. 3 mL of 100% methanol was added, and the mixture was vortexed vigorously until the precipitation had completely dissolved in methanol. A visible spectrometer (V-730 UV-Vis Spectrophotometer, Jasco, USA) was used to measure absorbance at 538 nm. The extracts' TTC was calculated as mg of linalool per gram of dry weight, or DW, of extract. The standard curve's equation was y = 0.0036 x 0.001, with R2 = 0.9927.
Determination of total Carotenoid
The study adopted the procedure previously described by[42] for quantification with modification. The plant materials (100mg) were ground and then extracted with methanol for 24 hours in continuous extraction (Soxhlet). The extraction was filtered, and methanol was evaporated in the rotary evaporator under vacuum at a temperature of 45 oC to dryness. Chemicals and solvents used in this assay were analytical grade and were acquired from POCH (Gliwice, Poland). To precipitate carotenoids, approximately 1.5 g of the sample was treated with Carrez I and II solutions. Afterwards, the extraction was carried out using acetone and petroleum ether. The absorbance value of the ether extract was measured at 450 nm. The standard solution was prepared by mixing 560µL of tool with lutein, ꞵ-cryptoxanthin, lycopene, 250µL of carotene, and 1.00 mL of phytoene stock solution, then diluting to 5000µL with 50:50 ethanol and acetonitrile. Then, from this, stock solutions of 1.5, 0.3, 0.6, 1.2, 1.5, 1.8, 2.1, and 2.4 mg/mL were prepared. It was determined from calibration curves obtained from the standards.
Determination of total Glycosides
The procedure previously described by[43] for quantification was adopted with modifications in the standard solution of luteolin-7-glycoside. The plant materials (100mg) were ground and then extracted with methanol for 24 hours in continuous extraction (Soxhlet). The extraction was filtered, and methanol was evaporated in the rotary evaporator under a vacuum at a temperature of 45 o C to dryness. 10 mL of freshly made Baljet's reagent (95 mL of 1% picric acid and 5 mL of 10% NaOH) was combined with a 10% extract of each generation and a total extract of seeds to determine the presence of cardiac glycosides. In order to test the absorbance at 495 nm, the mixture was diluted with 20 mL of distilled water after an hour. A 1.0 mLtandard solution of luteolin-7-glycoside was dissolved in a 25- mL flask with 2 mL of a 2% aluminum chloride solution to create the luteolin-7-glycoside. Different quantities of 1.5, 0.3, 0.6, 1.2, 1.5, 1.8, 2.1, and 2.4 mg/ mL were generated in this stock solution, and absorbance was measured at 495 nm to quantify these amounts of glycosides in the sample.
Determination of total Saponin
The method for quantification originally reported by[44], was used, although with changes in the standard solution of anisaldehyde reagent. The plant material (100 mg) was crushed before being continuously extracted with methanol for 24 hours (Soxhlet). The extraction was filtered, and methanol was dried by rotary evaporation at a temperature of 45 oC while being vacuum-sealed. 1.11 g of the natural sample (or 1.11 g of the corresponding dry weight for processed samples) was mixed with 10 mL of 50% ethanol to extract the saponins, which were then allowed to macerate for 72 hours at room temperature. The extracts were then filtered into 10-milliliter volumetric flasks and finished with 50% ethanol. 7 mL of Lieberman-Burchard reagent (16.7% of acetic anhydride in concentrated sulfuric acid) was mixed with 2 mL of the diluted extract (1:25 dilution), or standard, to complete the analysis. The solution was vortexed and scanned at 528 nm after standing for 30 minutes at room temperature. The dry material was quantified using a standard saponin curve (50–350 g/mL), and the findings were reported as mg/g.
Statistical Analysis
All the data was analyzed by GraphPad Prism® version 9.0.0. The Shapiro-Wilk test revealed that all the photochemical constituents were not normally distributed (p≥0.05) except saponin in the aqueous extract (p≤0.0194). The Kruskal-Wallis test was used to test for significant medium concentration differences among the same compound and different groups of compounds, and finally, the post-hoc test (Mann-Whitney tests) was used to locate the medium significant difference. Significant differences for saponins were determined by an independent student t-test. Data were expressed as mean ± standard deviation and summarized in tables in the following proceeding. Graphs were generated by the GraphPad prism.