INTRODUCTION:

Endophytic fungi are microscopic fungi that reside inside the tissues of plants without inducing disease symptoms¹. Plants have a lot of root endophytes. Host plants and endophytic fungi interact in a variety of ways, from mutualism to parasitism².

Schulz et al. 1999 proposed a "balanced antagonism" to define the endophyte-host interaction³. This is the equilibrium between fungal virulence and plant defenses. However, an imbalance in food exchange⁴, environmental factors⁵, and physiological stress, such as senescence⁶, may disrupt this equilibrium. As a result, Sieber 2002 defined endophytic fungi as those discovered in tissues that appeared to be healthy and functional at the time of sampling⁷. Fungal endophytes have been found in almost every plant species⁸. Endophytes produce a variety of medicinal compounds that are native to the host⁹. The majority of endophyte-produced chemical compounds have great economic value and are utilized to treat a variety of serious ailments, including cancer, inflammation, diabetes, and bacterial and fungal infections¹⁰.

Jatropha variegata belongs to the family: Euphorbiaceae a Yemeni plant species¹¹. Similar to other Jatropha species, it has been used in folk medicine in Yemeni to treat wounds^12,13, and as a contraceptive¹⁴. Antitumor, antibacterial, anticoagulant, antioxidant, antiprotozoal, immunomodulating, anti-inflammatory, insecticidal, and wound healing properties have been discovered in Jatropha species¹⁵. Terpenes, coumarins, alkaloids, ester ferulates, cyclic peptides, phloroglucinols, flavonoids, lignans, coumarino-lignoids, a noncyanogenic glucoside, fatty acids phenolics, and deoxypreussomerins are among the bioactive components found in Jatropha species¹⁶.

On the other hand, the plant Acokanthera schimperi, which grows in southern Yemen and belongs to the Apocynaceae family, is frequently used for the treatment of wounds¹⁷ and microbiological illnesses such as bacterial infection of the nails, tonsillitis (leaf), sexually transmitted diseases and leprosy (leaf). It's also used to cure scabies, dermatitis (leaf), rheumatic pain (stem), swelling (root and leaf), headache (root and bark), elephantiasis (root), common cold (leaf), and rheumatic pain (stem)¹⁸. Acokanthera schimperi possesses a range of secondary metabolites, including alkaloids, flavonoids, coumarin, cardiac glycosides, terpenes, anthraquinone, tannin, and phenolic compounds, according to phytochemical investigations¹⁹.

Ceropegia rupicola, a member of the Apocynaceae family, is native to Africa, southern Asia, and Australia ²⁰. Externally administered for skin problems, commonly known as "Bukira." Ceropegia species contain a wealth of valuable phytoconstituents such as albuminoid, starch, gum, fats, sugars, crude fiber, and other valuable phytoconstituents that are commonly used in traditional Indian ayurvedic medicines for the treatment of gastric disorders, diarrhea, dysentery, and urinary tract disorders²¹. The species is extremely rare, and no research has been done on it yet.

In this study, three plant species, Acokanthera schimperi, Ceropegia rupicola, and Jatropha variegate were studied to the isolation of endophytic fungus and the antibacterial and cytotoxicity activities of their methanolic extracts. The phytochemical makeup of three plant extracts was also investigated.

MATERIALS AND METHODS:

Materials:

Materials of plant:

Acokanthera schimperi, Ceropegia rupicola, and Jatropha variegate were the plants employed in this investigation. In the spring (March – April), they were harvested by Prof Dr/ Abdu Ghalib AL Kolaibe of the Microbiology Department, Faculty of Science, Taiz University, Taiz, Yemen. The plant components were collected and then shade dried at room temperature before being baked (35^oC). A grinder was then used to grind the dry plant ingredients.

Microorganisms:

The screening technique for detecting antibacterial activity included eight microorganisms: - Gram-positive Staphylococcus aureus ATCC 6538, Staphylococcus mutants, Bacillus cereus ATCC 6633017, and Gram-negative Pseudomonas aeruginosa ATCC 9027015, Escherichia coli NCTC 10418012, Klebsiella pneumoniae ATCC 13883015, Vibrio cholera ATCC 700012, and Yeast Candida albicans ATCC 700012.

Media:

Starch casein medium²², potato dextrose agar medium²³, and nutrient agar medium (DEFCO Laboratories, USA) were all obtained from the Department of Chemistry of Natural and Microbial Products, Pharmaceutical Industries Researches institution, National Research Centre, Giza, Egypt.

Methods:

Isolation of endophytic fungi:

Surface sterilization of samples followed the standard approach^24-25 to eliminate undesired fungal propagules clinging to plant components. The upper, middle, and bottom portions of leaf and stem samples from the three studied plants were cleaned under running water and cut into 5 x 5mm pieces with a flame-sterilized blade. Using sterile filter paper, surplus moisture was wiped. In 6cm diameter Petri-dishes with Potato dextrose agar medium, the surface-sterilized materials were equally distributed (modified with streptomycin 150mg/l). The Petri plates were sealed with parafilm and kept in an incubator at 27°C for four weeks. The Petri-dishes examined endophytic fungal colonies that grow from the segments daily. The hyphal tips that developed out of the segments were separated and sub-cultured onto PDA before being introduced to culture pure. All endophytic fungi isolates were kept in PDA slants and documented.

Morphological examination of endophytic fungi:

The fungi were identified using Kong and Qi's methodology²⁶ based on morphological traits. Observations were used to create colony descriptions by using a PDA under natural light. After 72 hours, rates of growth at 20, 25, 30, 35, and 40^oC have been determined using published techniques²⁷. Preparations mounted in lactic acid were used to make microscopic observations and measurements. During conidia maturation, which takes 4–7 days, macronematous conidiophores were taken from the borders of conidiogenous fascicles or pustules to study morphology and conidiophore structure.

Preparation of extracts:

The plant parts were dried outside in the open air, away from direct sunlight, in all portions (leaf and stem). Separately, the dried plant elements were pulverized to the appropriate size. Each powdered plant material was extracted for 48 hours in 96% methanol (500ml) with extra agitation and the extracts were filtered through filter paper Whatman No. 3 (Whatman Ltd., England) at the end of the extraction period. Three times (1:1 (v/v)) extraction was performed on the three plants. The organic solvents were extracted by evaporation at 40^oC using a rotatory evaporator (Bauchi, England). The water was subsequently removed from the aqueous residues by baking them at 40°C for 48 hours. Each plant's dried bulk was then pulverized, placed into a glass vial, and kept refrigerated until needed.

Assay of antimicrobial:

The disc diffusion technique was used to test the extracts for antimicrobial activity. The nutrient agar medium was made by dissolving 27g/l of nutrient agar in water. Sterile nutrient agar inoculated was placed onto sterile Petri-dishes containing microbial cells of the microorganisms under test (200mL suspension of each microbial cell in 20mL agar medium). 20ml of each of the three distinct extract solutions of Acokanthera schimperi, ceropegia rupicola, and Jatropha variegate were impregnated into sterile filter paper discs with a diameter of 6mm (equal to 4mg of dry extract). After allowing the paper discs to dry at room temperature, they were placed on top of the inoculated agar plates. To allow the extracts to diffuse into the agar, the plates were placed in refrigerated for 2 hours. The plates were then incubated for 18 hours at 37°C²⁸. Negative control discs were made by immersing them in a methanol solution with 20 L of methanol. Positive controls included erythromycin and ciprofloxacin discs. MeOH was used to saturate paper discs with 10L of 0.25 mg/mL of ciprofloxacin and erythromycin²⁹. The inhibition zones were used to assess the antimicrobial activity at the end of the incubation period (the size of the inhibition zone plus the diameter of the disc).

The Neutral red uptake (NRU) assay (cytotoxicity measurement):

Cell culture:

HCT-116 (colorectal carcinoma) was kept in RPMI medium, whereas In DMEM media, A549 human lung carcinoma and Pc3 (prostate cancer) were grown. Also, in DMEM F12 media, BJ-1 (human normal immortalized skin fibroblasts) was grown. All of the media were supplemented with 10% fetal bovine serum before being incubated at 37°C, 5% CO₂, and 95% humidity. The cells were sub-cultured with 0.15 percent trypsin versine. Professor Stig Linder, Oncology and Pathology department, Karolinska Institute, Stockholm, Sweden, kindly provided an immortalized normal foreskin fibroblast cell line (BJ-1). Also, additional cell lines were donated by VACSERA (Giza, Egypt).

Cell viability assay:

After 24 hours of seeding 20000 cells per well in 96 well plates for A549, PC3, HCT-116, and BJ-1, the media was changed to a serum-free medium with a final concentration of the extracts of 100g/ml in triplicates. The cells were given a 48-hour treatment. Positive control of 100 g/ml doxorubicin was employed, while a negative control of 0.5% DMSO was used. To assess cell viability, MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was utilized³⁰.

The formula for calculating % cytotoxicity:

(av(x) / (av (NC))) *100

Where Av stands for average, X for sample well absorbance recorded at 595nm with a reference of 690 nm, and NC for negative control absorbance measured at 595nm with a reference of 690nm.

Determination of IC50 values:

Different concentrations of highly active samples with a strong cytotoxic effect on a variety of cancer cell lines were generated for dose-analysis experiments. The IC50 values of each sample were computed using probit analysis and the SPSS computer program (SPSS for Windows, statistical analysis software package/version 9 / 1989 SPSS Inc., Chicago, USA).

Analysis via GC-MS (Gas Chromatography-Mass Spectrometry): -

At Research City in Alexandria, Egypt, the chemical content of each of the three plant extracts was analyzed using GC-MS. The GC-MS analysis was carried out on a Shimadzu GCMS-QP2010 SE system (Kyoto, Japan) with a flame ionization detector (FID), an RTx-5 capillary column (dimensions: 30m 0.25mm, film thickness: 0.25m), and a split-spitless injector in EI mode (70 eV). The temperature of the starting column was set at 50°C for 3minutes (isothermal), then scheduled to increase to 200°C at an average of 15°C every minute for 5 minutes (isothermal). The temperature was then designed to rise to 240°C at an average of 3°C per minute, then stay at that degree for 10minutes (isothermal). Finally, the temperature was set to rise at a rate of 4°C per minute until it reached 300°C before remaining at that temperature for 10 minutes (isothermal). The injector and detector were both kept at a constant 280°C temperature. For 2 minutes, the oven temperature was set at 45°C, then gradually increased to 300°C at a rate of 5°C/min for 5 minutes before remaining at that temperature. Diluted samples (1% v/v) were automatically injected in split mode using a Shimadzu autoinjector and helium as a carrier gas at a flow rate of 1.41ml/min (split ratio 1: 15). All mass spectra were recorded using computerized integration. The measurements were performed with the following parameters: 60mA filament emission current, 70eV ionization voltage, and a 220°C ion source.

RESULTS:

Isolation and identification of endophytic Fungi:

Endophytic fungi have been isolated from 80 segments from three plants (Acokanthera schimperi, ceropegia rupicola, and Jatropha variegate), including leaves (50) and stem (30) samples. From these segments, 40 cultures were obtained. The findings revealed that the frequency of colonization varied greatly. As shown in Table (1), the rate of colonization was highest in samples of the leaves (60%) and lowest in samples of the stem (34%).

Table (1): - The rate of colonizing endophytic fungi isolated from three plants under examinations

Isolation site	No. of analyzed segments	No. of endophytes	Colonization rate (%)
Leaf	50	30	60
Stem	30	10	34
Total	80	40

On the other hand, the 40 isolates of fungal endophytes that were obtained on starch casein and potato dextrose agar media were identified based on microscopic features, and the results were as follows: As indicated in Table (2), 15 belonged to Fusarium spp., 6 isolates to Phoma spp., 5 isolates to Alternaria alternata, 6 isolates to White sterile mycelia, 6 isolates to Dark sterile mycelia, 1 isolation to Cochliobolus lunatus, and 1 isolate to Mucorspp (2). Figure(1) also depicts the morphology of various isolates (i.e., spore properties and fruiting structure).

Table (4): The antimicrobial activity of three methanolic extracts of Jatropha variegate, Ceropegia rupicola, and Acokanthera schimperi

			Inhibition zone (mm)
Code	Microbial strains		*Jatropha variegata*	*Ceropegia rupicola*	*Acokanthera schimperi*
1	Gram-negative bacteria	Vibriocholera ATCC 700012	-	11	15
2		Pseudomonas aeruginosa ATCC 9027015	-	13	8
3		Escherichia coli NCTC 10418012	15	-	10
4		Klebsiella pneumonia ATCC 13883015	45	10	15
5	Gram-positive bacteria	Bacilluscereus ATCC 6633017	10	8	12
6		Staphylococcusaureus ATCC 6538	-	-	-
7		Staphylococcus mutants	-	-	-
8	*Yeast*	Candidaalbicans ATCC 700012	23	20	18

Table (5): Cytotoxicity activity of Jatropha variegate, Ceropegia rupicola and Acokanthera schimperi against different cancer cell lines

Sample Type	Cytotoxicity % at 100 ug/ml			Normal cell line (BJ-1) Human normal immortalized skin fibroblasts
Sample Type	A549 Human lung carcinoma	PC3 Prostate cancer	HCT-116 Colorectal carcinoma
*Jatropha variegate*	59.2	94	74.3	95.8
*Acokanthera schimperi*	32.4	38.3	27.5	16.6
*Ceropegia rupicola*	91	90.7	94.7	92.2
Doxorubicin (Positive control)	99.5	98.6	100	98.7

Table (6): The IC₅₀ of the most effective plant extracts on the cancer cell line PC3 and A549 and normal cell line Bj-1

Sample code	IC₅₀ug/ml
Sample code	PC3 Prostate cancer	A549 Human lung carcinoma	Bj-1 Human normal immortalized skin fibroblasts
*Jatropha variegate*	39.4	84.1	59.9
*Ceropegia rupicola*	34.0	49.4	42.5

	A549	PC3	HCT-116	Bj-1
1
2
3
Untreated cells

Figure (2): - The morphological effects of the plant extracts examined on several cancer cell lines

Gas chromatography-mass spectrometry (GC-MS) analysis: -

To identify distinct compounds inside a sample, scientists utilize a technique that combines mass spectrometry and gas chromatography (GC-MS). It works by using GC to separate different compounds based on their RT and then using MS to study the separated compounds at a molecular level. Table (7) shows the presence of 13 phytoconstituents discovered by GC-MS analysis of Jatropha variegate extract. The most abundant components were ethanol which showed the highest area (98.33 %), followed by 4-Hepten-3-one, 4-methyl- with area (0.670 %), oxirane methanol with area (0.390 %), formic acid, propyl ester with area (0.270 %), formic acid, ethyl ester and Furfural with area (0.150 %) for both, Acetic acid, hydroxy- with area (0.043 %), 4-Methyl-3,4-dihydro- [1,2,3] triazolo[4,5-d] pyrimidine-5,7-dione with are (0.010%). On the other hand, compound glycidol, acetic acid, hydroxy-, ethyl ester hydrazine, methyl and oxirane, 2,3-dimethyl-are fragments, that are found in low intensity, for that it can be only identified by GC-Ms but not quantified.

In addition, analysis of GC-MS of the Acokanthera schimperi extract proved the presence of 12 phytoconstituents, which are listed in Table (8). Formic acid was the most abundant component, accounting for 31.31% of the total area, followed by acetic acid, hydroxy-, ethyl ester, and acetic acid, hydroxy- (22.16 and 22.13%, respectively), phthalic acid, 6-ethyl-3-octyl butyl ester (14.75%), and butanoic acid, 2-methyl (9.650%). Compound ethanol, methylamine, propiolactone, formic acid, propyl ester, hydrazine, methyl-, oxalic acid, cyclobutyl heptyl ester, and 2-propenoic acid, ethenyl ester are other fragments that are discovered in low concentrations and can only be detected by GC-Ms but not quantified. Furthermore, the analysis of GC-MS of Ceropegia rupicola extract proved the presence of only six phytoconstituents, as shown in Table (9). The most abundant components were ethanol with an areaof (99.97%), followed by dimethyl ether with an areaof (0.034%). On the other hand, compound acetic acid, hydroxy-, propanoic acid, 2-hydroxy-, ethyl ester, formic acid, ethyl ester, and ethanol, 2-nitro-, nitrate (ester) are low-intensity fragments that can only be recognized but not quantified by GC-Ms.

Table 8: Phytoconstituents of the methanol extract of Acokanthera schimperi

ID	RT	Compound name	M. F.	M. Wt.	Peak area%
1	-	Ethanol	C₂H₆O	184	-
2	4.473	Acetic acid, hydroxy-, ethyl ester	C₄H₈O₃	104	22.16
3	5.166	Acetic acid, hydroxy-	C₂H₄O₃	152	22.13
4	-	Methylamine	CH₅N	62	-
5	-	Propiolactone	C₃H₄O₂	288	-
6	-	Formic acid, propyl ester	C₄H₈O₂	176	-
7	-	Hydrazine, methyl-	CH₆N₂	322	-
8	-	Formic acid	CH₆N₂	230	31.31
9	15.221	Butanoic acid, 2-methyl	C₅H₁₀O₂	306	9.650
10	15.617	Phthalic acid, 6-ethyl-3-octyl butyl ester	C₂₂H₃₄O₄	362	14.75
11	-	Oxalic acid, cyclobutyl heptyl ester	C₁₃H₂₂O₄	484	-
12	-	2-Propenoic acid, ethenyl ester	C₅H₆O₂	98	-

Table 9: Phytoconstituents of the methanol extract of Ceropegia rupicola

ID	RT	Compound name	M. F	M. Wt.	Peak area%
1	3.083	Ethanol	C₂H₆O	368	99.97
2	-	Acetic acid, hydroxy-	C₂H₄O₃	228	-
3	3.840	Dimethyl ether	C₂H₆O	92	0.034
4	-	Propanoic acid, 2-hydroxy-, ethyl ester	C₅H₁₀O₃	118	-
5	-	Formic acid, ethyl ester	C₃H₆O₂	148	-
6	-	Ethanol, 2-nitro-, nitrate (ester)	C₂H₄N₂O₅	136	-

DISCUSSION:

Terpenoids, steroids, flavonoids, phenols, and alkaloids are plant-derived medicinal bioactive compounds utilized in the pesticide, pharmaceutical, and cosmetic industries ³¹. Three plant species were used in this investigation, namely Acokanthera schimperi, ceropegia rupicola, and Jatropha variegate were investigated for their isolation of endophytic fungi and most species of endophytic fungus have been described based on morphological criteria such as peridium morphology, color, ascospore, organoleptic properties, glebe scent, and other, according to classical mycology³². Endophytic fungal colonies are extremely common in tropical plant leaves³³. The current research findings back up this assertion. The current findings revealed that endophytic fungal colonization occurs more frequently in leaves than in stems, with leaves samples having the highest colonization frequency (60%). Other investigations in the same field³⁴ have shown similar results. Furthermore, the results revealed that 40 isolates of fungal endophytes were obtained, with Fusarium spp. being the most isolated strain (15 isolates). The plant species used, the isolation medium used, and the amount of sample used in this study are the most important potential effective parameters for the isolation rate of endophytic fungi.

We only employed two isolation media (potato dextrose and starch casein) and a limited number of samples which may be responsible for minimizing the isolation of endophytes. So, increasing the sample number and isolation media, as well as the investigation scope and rationale, may result in the recovery of additional endophytic fungi^35-36. The highest yields were obtained from Jatropha variegate (45.4% w/w), followed by Acokanthera schimperi (32.3% w/w), and finally Ceropegia rupicola (20.5% w/w) according to the results of methanolic extracts. Furthermore, our findings revealed that the three extracts had a stronger effect against Gram-negative bacteria (Vibrio cholera ATCC 700012, Escherichia coli NCTC 10418012, Pseudomonas aeruginosa ATCC 9027015, and Klebsiella pneumoniae ATCC 13883015) than Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 6633017, and Staphylococcus mutans). This does not agree with the theory that proved that Gram-positive bacteria are more sensitive than Gram-negative bacteria because Gram-negative bacteria have a more complex cell wall/membrane structure ^37-39. Gram-negative bacteria have more complex cell walls than Gram-positive bacteria, which acts as a diffusional barrier, rendering them less susceptible to antimicrobial drugs than Gram-positive bacteria^40,41. Gram-negative bacteria have extra complex cell walls than Gram-positive bacteria, which function as a diffusional barrier, reducing their susceptibility to antimicrobial medicines. The extract Acokanthera schimperi furthermore, proved activity against Gram-negative bacteria Bacillus cereus ATCC 6633017, which have a low activity (12 mm), and the phytochemical constituents of this plant indicate that it contains very potent cardiotonic, according to the literature review and the main compound of which is ouabain^42,43.

In addition, an Ethiopian investigation on A. schimperi methanolic proved the growth inhibition of E. coli, P. aeruginosa, and P. vulgaris⁴⁴. Furthermore, all three extracts were efficient against Candida albicans ATCC 700012, a species of yeast, which was a first for A. schimperi. On the other hand, the methanolic extract of Jatropha variegate has the strongest antibacterial action against Klebsiella pneumoniae ATCC 13883015. (45 mm). A prior study⁴⁵ found that the leaves extract had good antibacterial activity. Extraction temperature, duration, type of solvents used, and volume are all factors⁴⁶. Furthermore, the extract of Ceropegia rupicola was effective against Candida albicans ATCC 700012, Bacillus cereus ATCC 6633017, and most Gram-negative bacteria. This is owing to the genus Ceropegia's pharmacological importance, which stems from the presence of a pyridine alkaloid known as 'Cerpegin'^47,48.

Cancer is the deadliest disease in the world, and its prevalence is projected to rise as people adopt behaviour’s and lifestyle variables linked to cancer⁴⁹. The use of chemotherapeutic drugs in cancer treatment is currently limited by toxicity and tumor resistance. Natural goods are a fantastic way to test innovative and secure anti-cancer treatments⁵⁰. Bioactive compounds are abundant in plant extracts due to a diversity of chemical ingredients like polyphenols, alkaloids, and flavonoids, all of which play an essential role in drug development⁵¹.

As a result, a well-established assessment of these three plant extracts' anticancer potential was performed in this study to see how effective they are at stopping cell proliferation on three various human cancer cell lines: PC3, A549, and HCT-116, and the results revealed that the extract of Ceropegia rupicola had the highest effective anticancer activity against HCT-116, with 94.7 % cell growth inhibition, followed by extract Jatropha variegate, followed by extract Jatropha variegate against PC3 with 94% of cell growth inhibition. Then came PC3 and HCT-116, which inhibited cell proliferation by 91 and 90.7%, respectively. Acokanthera schimperi extract inhibited cell proliferation in A549, PC3, and HCT-116 cells at low levels (32.4, 38.3, and 27.5%), respectively. A methanolic extract of Ceropegia rupicola with an IC50 of 111 mg/ml was the most active extract and these results are according to Alasbahi and Melzig, 2008⁵¹. Newman and Cragg 2012 also discovered that the anticancer activity of the root of Aloe pirottae, which was evaluated against lung cancer (A549) cell lines, has an IC50 value of 29.69g/mL⁵². Furthermore, our results on the Jatropha variegate extract on A549 show high IC50 with values of 84.1 ug/ml, then normal cell line (Bj-1) with an average IC50 of 59.9 ug/ml, and finally the low value of IC50 at 39.4 ug/ml was recorded for PC3 cell line. Moreover, Ceropegia rupicola extract proved average IC50 at 49.4 and 42.5 for A549 and normal cell line (Bj-1) respectively, and the IC50 at 34 ug/ml for PC3 cell line. According to the US National Cancer Institute, raw extract can be inert, moderately active, or active in terms of cytotoxicity⁵³. Plant chemicals that include many components increase the amount of oxidative stress in cancer cells by suppressing inactivating pro-survival signals, generating DNA damage, inhibiting signalling pathways, and activating apoptosis-related signals that promote cancer cell growth⁵⁴.

GC-MS is extremely trustworthy because it extracts chemicals in their purest form. The ability of this tool, as well as the solvent, to produce more products. The use of GC-MS to extract chemicals can provide pharmaceutical businesses with a large platform on which to construct diverse drugs from plants that can be a good source of these drugs⁵⁵. Jatropha variegate extract of methanol contains thirteen primary ingredients, according to the phytochemical study using GC-Mass: - ethanol, 4-hepten-3-one, 4-methyl-, oxirane methanol-, formic acid, propyl ester-, formic acid, ethyl ester-, furfural, acetic acid, hydroxy-, 4-methyl-3,4-dihydro- [1,2,3] triazolo[4,5-d] pyrimidine-5,7-dione, glycidol, acetic acid, hydroxy ethyl ester-, hydrazine and methyl and oxirane, 2,3-dimethyl. The bioactive components range from ethanol, formic acid, propyl ester-, formic acid, ethyl ester-, furfural, and acetic acid. These primary phytoconstituents have been linked to antibacterial and anticancer capabilities, as evidenced by the results^56-57, and are similar to those found in other Jatropha species like J. glauca and J. curcas⁵⁸. The GC-MS examination of Acokanthera schimperi extracts, on the other hand, revealed the presence of twelve phytochemical compounds like formic acid, acetic acid, hydroxy-, ethyl ester, acetic acid, hydroxy-, phthalic acid, 6-ethyl-3-octyl butyl ester, butanoic acid, 2-methyl, ethanol, methylamine, propiolactone, formic acid, propyl ester, hydrazine, methyl-, oxalic acid, cyclobutyl heptyl ester, 2-propenoic acid, ethenyl ester and retention time (RT), peak area, molecular weight, and molecular formula were used to confirm their presence. Additionally, preliminary phytochemical screening assays may be effective in detecting bioactive principles, which could lead to medication discovery and development. Furthermore, these tests make it easier to estimate the quantity of pharmacologically active chemical substances and to separate them qualitatively ^59-60. Also, the GC-MS analysis of Ceropegia rupicola extracts proved that the existence of six compounds ethanol, dimethyl ether, acetic acid, hydroxy-, propanoic acid, 2-hydroxy-, ethyl ester, formic acid, ethyl ester and ethanol, and 2-nitro- nitrate (ester).

CONCLUSION:

Some medicinal plants used in Yemeni traditional medicine for wound and skin healing areAcokanthera schimperi, Ceropegia rupicola, and Jatropha variegate. These plants showed antibacterial activity against most Gram-negative bacteria, some Gram-positive bacteria, and yeast in this investigation.Also, exhibited cytotoxicity against A549, PC3, and HCT-116. The presence of ethanol, formic acid, acetic acid, methylamine, propiolactone, propanoic acid, oxalic acid, furfural, phthalic acid, and dimethyl ether chemicals in the phytochemical screening of the three plants appears to contribute to the antibacterial and cytotoxicity capabilities. The study confirmed that Acokanthera schimperi, Ceropegia rupicola, and Jatropha variegate are all promising medicinal plants, but additional studies are needed to prove their effects.

CONFLICTS OF INTEREST:

The authors declare that there is no conflict of interest

ACKNOWLEDGMENTS:

The authors acknowledge the National Research Centre (NRC) of Egypt for providing all needed facilities and logistics for the study. Also, thanks to Prof Dr/ Abdu Ghalib AL Kolaibe of the Microbiology Department, Faculty of Science, Taiz University, Taiz, Yemen for supporting the plants.

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Received on 16.07.2022 Modified on 22.08.2022

Research J. Pharm. and Tech 2023; 16(4):1833-1842.

DOI: 10.52711/0974-360X.2023.00301

Plant spp.	Part extracted	yield % (w/w) (average ± S.D.)
*Acokanthera schimperi*	Leaf + stem	32.3 ± 2
*ceropegia rupicola*	Leaf + stem	20.5 ± 3
*Jatropha variegate*	Leaf + stem	45.4 ± 2