Exploring polyphenolic profiles by using a mutlvariate approach in Citrullus colocynthis (L.) Schrad

Abstract

Plants have held profound cultural and medicinal significance throughout human history. Citrullus colocynthis (L.) Schrad, known as Tumba in Rajasthan, Kaudtumba in Punjab, and Garhtumba or Indriyian in Haryana, stands as a crucial wild plant, resembling the watermelon vine and thriving in arid terrains. Local communities have long utilized this wild plant for diverse purposes. The genus is abundant in secondary metabolites, playing a pivotal role in the body's defense mechanisms. This study aimed to unravel the essential phytochemicals present in Citrullus colocynthis. The preliminary phase involved quantitative analysis, followed by screening of total phenolic and flavonoid content of different accessions. Subsequently, HPLC was employed to quantify the variance of phenolics and flavonoids in 10 selected accessions. The study highlights significant phenolic content in accession CRR19064 (69.17 ± 0.03 mg TAE/g) and CRB19004 (69.12 ± 0.01 mg TAE/g) from methanolic extracts. Regarding flavonoids, accession CHR19013 displayed the highest content (76.74 ± 0.03 mg QE/g), followed by accession CPM19021 (74.37 ± 0.01 mg QE/g) from methanolic extracts, underlining their potential medicinal benefits. The Principal component analysis (PCA) was used to explore correlations between bioactive components and accessions. The first five principal components explained 83.41% of cumulative variability. Further, Agglomerative Hierarchical Analysis categorized the accessions into four clusters. These findings crucial to discover the therapeutic potential applications of this wild plant, paving the way for future research and targeted utilization of its bioactive components. In conclusion, the study underscores the wealth of bioactive compounds in C. colocynthis, reaffirming its potential in traditional medicine and providing valuable insights for future pharmacological research and drug development.

Introduction

In India, people have a strong tradition of using resources wisely. Rural communities often worship plants and animals in their daily lives and traditions. Plants are highly respected and serve important roles in medicine, cooking, and caring for livestock, as highlighted in ancient texts like the Vedas. Traditionally, people have relied on nature for their food, medicine, and shelter. In developing countries like India, millions depend on natural resources for their daily needs and livelihoods. The appeal of medicinal properties in wild plants has led to a high demand, and the pharmaceutical industry heavily relies on them for valuable compounds. The Cucurbitaceae plant family, comprising the two sub-families, Cucurbitoideae and Zanonioideae, is widely used. Around 90% of traditional Indian remedies come from plants. Wild medicinal plants have been the preferred choice for daily tasks, especially for tribal communities [1]. Not only tribes but also 70% of the total population, including tribes, rely on natural medicines.

Traditional medicine therapists use around “1000” genera and “2500 “species for drug preparation. Recent studies show that about 75,000 plant species are used for medicinal purposes by ethnic groups. However, some plants, both wild and cultivated, used by tribes have not undergone enough clinical investigation. Scientists today are increasingly interested in studying these traditional plants for new insights. Tribal groups, with their long history of using wild plants, provide valuable information. Initially, indigenous people provided information about plant uses, which was then cross-checked with medical reviews to ensure practicality. One such plant is Citrullus colocynthis, n = 11, a wild perennial vine with pepo fruits used by tribes for a long time but not extensively studied scientifically [2]. Tribal knowledge, passed down orally, lacks written documentation. Despite this, tribes propagate the knowledge admirably. Gathering information from such communities and studying these plants scientifically is crucial and resourceful.

Morphologically, Citrullus colocynthis is well-adapted to dry conditions with features like lobular tendrils, rough stems, and yellow flowers [3, 4]. It serves various purposes, from treating ailments to cooking, providing income for arid region residents. Its phytochemical composition includes numerous active compounds, making it valuable for medicinal and economic purposes. This plant is used to treat various disorders like boils, pimples, constipation, and joint inflammation, as well as for cooking purposes [5–7]. However, the trend of clearing land for farming is threatening these plants, leading to the extinction of some on a daily basis. Conservation efforts are needed to protect these valuable plant species. C. colocynthis, renowned for its antiseptic and anti-diabetic properties, holds a prominent place in India for its various health benefits [8]. This study aims to determine the polyphenolic content and comparative HPLC profile of the fruit pulp of C. colocynthis, shedding light on its potential medicinal value.

Materials and methods

Plant material

Numerous areas across the North-Western plains of India, encompassing Haryana, Punjab, Rajasthan, and Uttar Pradesh, were selected for germplasm collection. Given the preference of the wild crop for arid terrain, collection visits were omitted in regions of Himachal Pradesh and Uttarakhand. The plant material was collected by Miss Navneet Kaur in month of April to June in 2017–2019 and identified by Dr. Manish Kapoor. The specimens have been submitted to the Herbarium at Punjabi University, Patiala. The acquired germplasm was subsequently cultivated in experimental plots at the Dr. S. S. Bir Botanic Gardens, situated within the Department of Botany at Punjabi University, Patiala. Detailed information regarding the visited locations and their respective global positioning system coordinates can be found in (Table 1). Additionally, a visual representation of these accessions can be seen in (Fig. S1).

Table 1. Table showing data of localities along their GPS coordinates for Citrullus colocynthis (L.) Schrad selected 10 Accessions

S.No	ID Number	Latitude	Longitude	Location	Altitude (m)
1	CRB19004	28.05° N	73.48° E	Bikaner, Rajasthan	97
2	CRG19007	29.86° N	73.79° E	Sri Ganganagar, Rajasthan	175
3	CHR19013	28.85° N	76.44° E	Rohtak, Haryana	222
4	CPM19021	30.0376° N	75.2931° E	Mansa, Punjab	212
5	CPM19030	30.3562° N	74.51° E	Sri Mukatsar Sahib, Punjab	198
6	CPB19036	30.2110° N	74.9455° E	Bathinda, Punjab	528
7	CRK19043	27.83° N	72.95° E	Bikaner, Rajasthan	217
8	CPA20064	31.8428° N	74.7630° E	Ajanala,Punjab	213
9	CPM20070	30.5887° N	76.8471° E	BhaiDesa, Mansa,Punjab	212
10	CRB20074	27.7966° N	73.3443° E	Deshnokh, Bikaner,Rajasthan	265

Sample extraction

A total of 10 morphologically diverse accessions were selected for fruit collection. To determine variability, principal component analysis and cluster analysis were applied to the data derived from 61 qualitative and quantitative morphological characteristics obtained from 78 accessions in the experimental plots. In the initial step, the fruits were carefully washed with tap water to eliminate any dust. Subsequently, the fruits were segregated into three parts: pulp, rind, and seed. These components were naturally shade dried and finely powdered using an electric blender. For the extraction process, 5 g of fruit pulp powder was dissolved in 100 mL of solvents. The extraction was conducted using various solvents, namely ethanol, methanol, petroleum ether, chloroform, and distilled water. The extraction process involved placing the powder in a shaker for a continuous 72-h period. After the extraction, the obtained extracts were filtered with Whatmann filter paper no. 4, and the filtered residues were dried using serological water bath at 40℃. The resulting dried residues were stored for future use in airtight containers at 4 °C [9].

Chemicals

HPLC-grade methanol was procured from Merck (Germany), while HPLC-grade chloroform, formic acid, and acetonitrile were sourced from Thermo Fisher Scientific (United States). Ultrapure water utilized in all experiments was produced using a Milli-Q Progard TS2 system (France). For HPLC analysis of phenols, the following standards from Sigma Aldrich were employed: caffeic acid, resorcinol, vanillic acid, coumaric acid, ferulic acid, vanillin, veratric acid, cinnamic acid, coumaric, pyrogallol, and various flavonoids including naringin, catechol, gossypin, luteolin, quercitin, apegenin.

Total phenolic content

Total Phenolic Content in the pulp of 10 morphologically diverse C. colocynthis accessions was determined using the Folin-Ciocalteu (FC) [10, 11]. Five solvents (methanol, ethanol, chloroform, petroleum ether, and distilled water) were used. The process involves conversion of phosphotungtic acid to phosphotungstic acid, a blue-colored substance causing absorbance increase. A 50 μL extract was mixed with 400 μL of 1N FC reagent and 3200 μL 20% Na₂CO₃ solution, followed by thorough shaking. 5 mL double distilled water was added, and after 2-h incubation at 20 °C, absorbance at 765 nm was measured. The absorbance of samples was compared to standard tannic acid, enabling calculation of standard tannic acid amount in each of the 10 accessions. The results were expressed as mg concentrations of total phenolic content per gram of dry fruit pulp, calculated using TAE (tannic acid equivalents). This screening procedure helped determine the optimal solvent for phenol measurement. The experiment was conducted thrice for precision.

Total flavonoid content

Flavonoids were quantified using the aluminium chloride colorimetric test [12]. The test solution was prepared using plant extract (fruit pulp), double distilled water, sodium nitrate, aluminium chloride, and sodium hydroxide. The concentrations used were 1000 μL of extract (1 mg/mL), 4000 μL, 0.3 mL of 50 g/L, 0.3 mL of 100 g/L, and 2000 μL of 1 M, respectively. Thorough mixing of all chemicals ensured proper amalgamation. Absorbance was measured at 510 nm, with quercetin as the standard. As a result, the data was reported as mg quercetin equivalent (QE) per gram of dry matter.

DPPH radical scavenging activity assay

The 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for the determination of the free radical scavenging activity of the all extracts to screen out best solvent [13]. For each extract and standard, sample solutions of different concentrations (2– 40 μg mL⁻¹) were prepared in methanol and added separately to an equal volume of 100 μM DPPH solution in methanol. The reaction mixture was kept at room temperature for 15 min. Then, the absorbance of the reaction mixture was recorded at 517 nm. Gallic acid was used as standard. Free radical scavenging activity was calculated using the following formula:$$\%\text{of free radical scavenging activity}=\left\{\left(\text{control OD}-\text{sample OD}\right)/\text{control OD}\right\}\times 100.$$

The extract concentration having 50% radical inhibition activity (IC₅₀) was calculated from the graph of the free radical scavenging activity (%) against the extract concentration. Three replicates were performed for each sample concentration to check the reproducibility of the experimental result and to get more accurate result. Results are represented as IC₅₀ ± standard deviation.

High performance liquid chromatography (HPLC) analysis

The methanolic extract of C. colocynthis underwent analysis to determine the content of 16 bioactive phenolic and flavonoid compounds. These included caffeic acid, resorcinol, vanillic acid, coumaric acid, ferulic acid, vanillin, veratric acid, cinnamic acid, coumaric, pyrogallol, and flavonoids such as naringin, catechol, gossypin, luteolin, quercitin, and apegenine. The analysis utilized an HPLC–DAD system. An Agilent Technologies Inc. HPLC system equipped with a column oven, binary gradient pump, autosampler, DAD detector, and shim-packed C18 column was employed for separation. Triplicate samples of the extracts, containing all 16 standard compounds (10 phenolic acids and 6 flavonoids), were meticulously prepared and filtered through 0.45 μm PVDF filters. An isocratic mobile phase of 55:45 (v/v) acetonitrile and phosphate buffer, along with an auto sampler, was used, and 10µL samples were injected into the column at 40 °C. After elution, the material was analyzed using a UV-based DAD detector at a wavelength of 280 nm, enabling the determination of the quantity of the 16 standards in the eluent. The retention time of sample peaks was compared to standard peaks, facilitating the quantification of sample substances. Results were presented as mg of each compound per g of dry weight (mg/g DW), and identification of compounds was achieved by comparing retention times and UV spectra of unknowns with standards [14].

Statistical analysis

All statistical analyses were performed using SPSS software package version 17. Data were expressed as the mean ± standard deviation. Data were compared by one-way ANOVA followed by the post-hoc LSD test [15]. Differences between groups were considered statistically significant when p < 0.05. XLSTAT was used on results attained from 10 Morphotype of C. colocynthis to do principal component analysis [16]. The goal of selecting the statistical technique was to precisely determine the correlation matrix, which leads to the contribution of each and every factor to genetic variation. Heat map matrices are also performed by online free SR Plot statistical software http://www.bioinformatics.com.cn/srplot.

Results

Total phenolic and flavonoids content

The total phenolic content was determined in 10 morphotypes of C.colocynthis using fruit pulp extracts from five different solvents: methanol, ethanol, chloroform, petroleum ether, and distilled water. The results were expressed as tannic acid equivalent (TAE). The tannic acid curve is depicted in (Fig. S2a). Analysis of the total phenol content data indicated significant differences among all ten accessions. Moreover, the solvents used (distilled water, ethanol, methanol, chloroform, and petroleum ether) showed significant variations. Methanol emerged as the most effective solvent for extraction. The highest phenolic content was reported in accession CRR19064 (69.17 ± 0.03 mg TAE/g), followed by CRB19004 (69.12 ± 0.01 mg TAE/g) from methanolic extracts. Conversely, the lowest phenolic content was recorded as 19.97 ± 0.03 mg TAE/g in accession CPA20074 in chloroform extracts, followed by accession CPM20070 with a phenolic content of 20.20 ± 0.02 mg TAE/g from Chloroform extracts.

The mean phenolic content values were highest for methanolic extracts (61.77 ± 0.02 mg TAE/g), followed by ethanol (52.69 ± 0.02 mg TAE/g), aqueous (47.65 ± 0.03 mg TAE/g), petroleum ether (31.73 ± 0.03 mg TAE/g), and chloroform (25.68 ± 0.02 mg TAE/g). Notably, accession CPA20074 showed the lowest phenolic content from methanolic extracts (48.43 ± 0.04 mg TAE/g), while for ethanol extracts, the minimum content was reported as 43.47 ± 0.03 mg TAE/g from CPM20070, followed by Aqueous 34.82 ± 0.01 mg TAE/g (CRG19007), Petroleum Ether 22.28 ± 0.03 mg TAE/g (CPA20074), and Chloroform 19.97 ± 0.03 mg TAE/g (CPA20074). CPA20074 exhibited significantly lower phenolic content compared to methanolic, petroleum ether, and chloroform extracts. Furthermore, the phenolic content varied significantly across accessions for different solvents.

The total flavonoid content was quantified in 10 morphotypes of C. colocynthis using fruit pulp extracts from five different solvents: methanol, ethanol, chloroform, petroleum ether, and distilled water . The results were expressed as quercetin equivalent (QE). The tannic acid curve is depicted in (Fig. S2b). In the analysis of the total flavonoid content, significant variations were observed among all ten accessions. Additionally, the solvents used ( distilled water, ethanol, methanol, chloroform, and petroleum ether) exhibited significant differences. Methanol was identified as the most effective solvent for extraction. The maximum flavonoid content was reported in CHR19013 (76.74 ± 0.03 mg QE/g) accession, significantly surpassing accession CPM19021 (74.37 ± 0.01 mg QE/g), followed by 74.1 ± 0.01 mg QE/g from methanolic extracts of accession CRB19004. Conversely, the minimum flavonoid content was recorded as 8.45 ± 0.03 mg QE/g in accessions CPA20074 and CRR19064 (8.45 ± 0.06 mg QE/g) from chloroform extracts, followed by accession CPM20070 with a flavonoid content of 10.98 ± 0.02 mg QE/g from chloroform extracts.

The mean flavonoid content values were highest for methanolic extracts (78.24 ± 0.02 mg QE/g), followed by ethanol (58.51 ± 0.03 mg QE/g), aqueous (43.77 ± 0.03 mg QE/g), petroleum ether (23.94 ± 0.03 mg QE/g), and chloroform (14.26 ± 0.02 mg TAE/g). Notably, accession CPA20074 showed the lowest flavonoid content from methanolic extracts (49.34 ± 0.04 mg QE/g), while for ethanolic extracts, the minimum content was reported as 31.36 ± 0.02 mg QE/g followed by aqueous 15.98 ± 0.03 mg QE/g (CPA20074), and petroleum ether 11.45 ± 0.06 mg QE/g (CPA20074). CPA20074 exhibited significantly lower flavonoid content compared to aqueous, petroleum ether, and chloroform extracts. Furthermore, the flavonoid content varied significantly across accessions for different solvents.

DPPH assay

The IC₅₀ (μg/mL) value indicates the effectiveness of a substance in inhibiting a specific biological or biochemical function. Lower IC₅₀ values indicate higher antioxidant activity because it takes a lower concentration of the substance to inhibit the process by 50%. The analysis of IC₅₀ values for different extracts reveals important insights into the antioxidant activity of various samples (Table 2). The sample CPA20074 demonstrates the highest antioxidant activity, as indicated by the lowest IC₅₀ values across most solvents. Specifically, for methanol, CPA20074 has an IC₅₀ value of 15.94 ± 0.03 μg/mL, for ethanol it was 23.94 ± 0.03 μg/mL, for water 71.97 ± 0.02 μg/mL, for chloroform 76.49 ± 0.02 μg/mL, and for petroleum ether, it is 87.49 ± 0.02 μg/mL. Additionally, sample CPM20070 shows the lowest IC₅₀ value for methanol, at 20.34 ± 0.02 μg/mL, indicating high antioxidant activity for this solvent. In contrast, the sample CRB19004 exhibits the lowest antioxidant activity, as reflected by the highest IC₅₀ values across all solvents. For methanol, the IC₅₀ value is 66.21 ± 0.01 μg/mL, for water, it is 153.87 ± 0.02 μg/mL, for ethanol, it is 86.43 ± 0.03 μg/mL, for chloroform, it is 140.28 ± 0.01 μg/mL, and for petroleum ether, it is 161.59 ± 0.03 μg/mL. These high IC₅₀ values suggest that a higher concentration of CRB19004 is required to achieve 50% inhibition of the DPPH free radicals, indicating lower antioxidant activity. The sample CPA20074 demonstrates the highest antioxidant activity across all tested solvents, as evidenced by its consistently low IC₅₀ values. Conversely, CRB19004 shows the lowest antioxidant activity with the highest IC₅₀ values.

Table 2. Antioxidant activity (DPPH) of pulp of Citrullus colocynth is expressed as IC₅₀

DPPH (%) IC50/μg/mL
Acc.no	Methanol	Ethanol	Water	Chloroform	Petroleum ether
CPA20074	15.94 ± 0.03	23.94 ± 0.03	71.97 ± 0.02	76.49 ± 0.02	87.49 ± 0.02
CPM20070	20.34 ± 0.02	24.41 ± 0.01	70.74 ± 0.02	84.12 ± 0.03	92.13 ± 0.01
CRR19064	49.14 ± 0.01	75.61 ± 0.02	139.72 ± 0.03	139.63 ± 0.02	149.82 ± 0.03
CPM19043	52.36 ± 0.02	81.34 ± 0.01	91.38 ± 0.02	97.49 ± 0.03	102.19 ± 0.01
CPF19036	46.38 ± 0.03	68.17 ± 0.02	107.43 ± 0.03	112.01 ± 0.01	131.29 ± 0.03
CPM19030	22.82 ± 0.01	29.34 ± 0.02	87.79 ± 0.03	84.29 ± 0.03	96.13 ± 0.01
CPM19021	56.28 ± 0.03	72.46 ± 0.03	107.17 ± 0.02	114.74 ± 0.02	129.92 ± 0.02
CHR19013	39.64 ± 0.02	48.61 ± 0.02	115.75 ± 0.03	112.18 ± 0.03	132.46 ± 0.02
CRG19007	52.14 ± 0.01	62.19 ± 0.01	103.49 ± 0.01	109.78 ± 0.02	114.01 ± 0.01
CRB19004	66.21 ± 0.01	86.43 ± 0.03	153.87 ± 0.02	140.28 ± 0.01	161.59 ± 0.03

Results are means ± SD of three independent experiments (n = 3) and statistically significant differences (p < 0.05). The Kruskal–Wallis H test indicated that there is a non-significant difference in the dependent variable between the different accessions

High Performance Liquid Chromatography

Caffeic acid, resorcinol, vanillic acid, coumaric acid, ferulic acid, vanillin, veratric acid, coumarin and cinnamic acid, naringin, catechol, gossypin, luteolin, quercitin and apegenin are few compounds among the most important secondary metabolites earlier reported from C. colocynthis pulp. Germplasm of different locations was analysed to find out the exact amount of these active constituents using HPLC. The content was expressed as (mg/g) on dry weight basis. Among the all standards used, retention time of apegenin was 25.077 min which was the maximum time taken to generate peak, while minimum was reported with caffeic acid, 4.235 min (Fig. S3a and S3b). Results demonstrated that a considerable amount of variations in phytoconstituents is reported in studied accessions. Value of caffeic acid varies from 66.657 caffeic acid mg/g to 40.0547 caffeic acid mg/g with mean value of 53.00249 caffeic acid mg/g. The value of pyrogallol was as low as 0.0125 in accessions CRB19004 to pyrogallol mg per gram. The highest content 66.657 caffeic acid mg/g was detected in CRB19004 with lowest 0.0125 content in CRB19004 (Tables 3, 4 and Fig. S3, Fig. S4).

Table 3. Concentration of phenolic compounds (mg/g) from fruit pulp of Citrullus colocynthis(L.)Schrad selected 10 Accessions

Accessions	Caffeic acid	Resorcinol	Vanillic acid	Coumaric acid	Ferulic acid	Vanillin	Veratric acid	Cinnamic acid	Coumaric	Pyrogallol
CRB19004	66.65 ± 2.83	7.45 ± 1.74	20.65 ± 1.12	13.88 ± 1.90	10.62 ± 2.17	4.69 ± 0.72	47.63 ± 1.52	0.77 ± 1.35	0.32 ± 1.72	0.01 ± 0.82
CRG19007	45.82 ± 1.89	4.25 ± 0.98	18.76 ± 0.82	10.49 ± 1.30	5.879 ± 2.11	1 ± 0.4093 ± 3.13	38.71 ± 1.35	0.63 ± 1.55	0.34 ± 0.85	0.53 ± 0.53
CHR19013	46.5 ± 1.35	3.62 ± 1.45	17.72 ± 0.85	18.34 ± 1.46	10.59 ± 0.74	2.81 ± 0.10	31.38 ± 1.62	0.70 ± 0.88	0.53 ± 0.94	0.02 ± 2.36
CPM19021	58.59 ± 1.9	2.16 ± 1.20	23.19 ± 1.42	15.84 ± 1.28	8.21 ± 1.23	1.16 ± 2.33	40.52 ± 1.56	0.74 ± 1.15	0.66 ± 0.76	0.02 ± 1.43
CPM19030	56.49 ± 3.16	6.24 ± 1.05	24.71 ± 0.78	18.59 ± 0.35	10.48 ± 1.56	3.24 ± 0.66	44.53 ± 1.23	0.66 ± 2.24	0.164 ± 1.38	0.03 ± 0.78
CPF19036	45.59 ± 0.96	5.32 ± 0.74	16.85 ± 0.89	14.92 ± 1.23	13.12 ± 0.78	1.78 ± 0.09	35.99 ± 0.66	0.43 ± 0.18	0.24 ± 1.67	0.01 ± 0.73
CPM19043	65.41 ± 1.15	7.15 ± 1.25	20.76 ± 1.32	14.08 ± 2.05	12.14 ± 1.34	3.99 ± 0.62	47.31 ± 1.24	0.48 ± 1.34	0.89 ± 1.58	0.02 ± 1.65
CRR19064	51.54 ± 1.88	5.32 ± 0.87	27.04 ± 0.66	18.57 ± 0.74	15.26 ± 0.89	1.09 ± 0.40	50.48 ± 3.80	0.49 ± 0.74	0.120 ± 0.52	0.31 ± 3.21
CPM20070	55.35 ± 1.44	3.62 ± 1.12	12.51 ± 0.58	11.05 ± 1.92	9.84 ± 3.21	2.85 ± 3.50	40.01 ± 1.56	0.94 ± 0.13	0.17 ± 0.45	0.05 ± 0.77
CPA20074	40.05 ± 0.93	5.29 ± 2.13	25.65 ± 0.83	12.67 ± 0.31	10.82 ± 3.82	4.58 ± 1.65	36.75 ± 2.02	0.83 ± 0.77	0.13 ± 1.72	0.02 ± 0.50

Results are means ± SD of three independent experiments (n = 3) and statistically significant differences (p < 0.05). The Kruskal–Wallis H test indicated that there is a significant difference in the dependent variable between the different groups.

The Post-Hoc Dunn's test using a Bonferroni corrected alpha of 0.0011 indicated that the mean ranks of the following pairs are significantly different: CRB19004 found significantly different to CRG19007, CPF19036, CRR19064, CPM20070 and CPA20074. CRG19007 significantly different to CPM19043. CHR19013 is found significantly different to CRR19064, CPM20070 and CPA20074. CPM19021 significantly different to CPM20070 and CPA20074. CPM19030 found significantly different to CPA20074. CPF19036 significantly different to CPM19043. CPM19043 significantly different to CRR19064, CPM20070 and CPA20074

Table 4. Concentration of flavonoid compounds (mg/g) from fruit pulp of Citrullus colocynthis(L.) schrad selected 10 accessions

Acc no	Naringin	Catechol	Gossypin	Luteolin	Quercitin	Apegenin
CRB19004	18.62 ± 0.09	43.65 ± 0.55	0.141 ± 1.23	0.15 ± 0.68	0.32 ± 0.06	ND
CRG19013	20.68 ± 1.12	47.63 ± 0.79	0.81 ± 0.88	0.13 ± 0.27	0.21 ± 0.09	60.40 ± 0.66
CHR19021	23.82 ± 0.89	23.71 ± 1.30	2.39 ± 1.71	0.10 ± 0.77	0.31 ± 0.56	66.57 ± 1.25
CPM19021	14.11 ± 0.55	30.96 ± 0.74	2.33 ± 0.92	0.20 ± 0.68	0.52 ± 1.15	83.49 ± 0.17
CPM19030	19.52 ± 2.32	40.81 ± 1.33	2.12 ± 1.65	0.25 ± 1.33	0.58 ± 1.09	81.86 ± 0.55
CPF19036	10.84 ± 0.79	38.82 ± 0.88	1.96 ± 1.23	0.18 ± 1.12	0.34 ± 0.77	45.67 ± 1.85
CPM19043	23.28 ± 1.43	55.74 ± 0.09	2.89 ± 0.89	0.69 ± 0.50	ND	84.57 ± 1.67
CRR19064	22.50 ± 0.27	57.25 ± 1.09	2.30 ± 0.74	0.64 ± 0.58	0.019 ± 1.04	76.65 ± 0.08
CPM20070	21.43 ± 0.58	56.35 ± 0.77	2.47 ± 3.34	0.57 ± 2.04	ND	66.93 ± 0.85
CPA20074	14.67 ± 0.98	39.40 ± 0.65	0.55 ± 0.93	0.49 ± 0.31	ND	56.95 ± 0.79

ND Not Determined, Results are means ± SD of three independent experiments (n = 3) and statistically significant differences (p < 0.05). The Kruskal–Wallis H test indicated that there is a non-significant difference in the dependent variable between the different groups

Based on the observed phenolic concentration, caffeic acid, veratric acid, and vanillic acid were found in substantial quantities. Conversely, coumaric acid, ferulic acid, resorcinol, and vanillin were present in moderate amounts. In contrast, cinnamic acid, coumaric acid, and pyrogallol were present in minimal amounts. In comparison to phenols, flavonoids were observed in higher concentrations. Within the flavonoid group, naringin and catechol were the most abundant, being highest among all the studied accessions. Following closely, quercetin was also found in notable amounts. On the other hand, gossypin, luteolin, and apigenin were present in the least amounts among the flavonoids.

The enhancement score in (Fig. S5) serves as a graphical representation illustrating the extent of variation in phenolic and flavonoid concentrations among the different accessions. This score allows for a quick and intuitive assessment of the performance of each accession concerning phenolic levels. In this particular case, CRB19004 and CPM19043 stood out as the accessions with the highest phenolic content, indicating their excellence in accumulating phenols. Conversely, CPF19036 displayed the lowest phenolic content, positioning it as the least proficient accession in terms of phenolic concentration. The variation in flavonoid content across the different accessions was relatively consistent, showing minimal variability. However, among the accessions, CRG19013, CRR19064, and CPF19036 emerged as the top-performing accessions, displaying the highest levels of flavonoids. Conversely, CPM19043 exhibited the least amount of flavonoids among the studied accessions. The ranking of accessions based on phenolic and flavonoid concentration provides valuable insights for further analysis and understanding of the phenolic composition in these plant accessions.

Evaluation of accession on the basis of variation of phenolic and flavonoids by Principal Component Analysis (PCA)

The concentration of various standards was chosen for PCA across ten different germplasms. PCA, a crucial tool for data analysis and fact-finding, was employed to examine the variation among C. colocynthis accessions collected from diverse localities in the plains of North-West India. Eigenvalues, variability percentages, cumulative percentages, and grouping of C. colocynthis accessions were computed based on the selected concentrations of different standards (Table 5). The outcomes were utilized to calculate the distance biplot, scree plot, and Pearson correlation matrix, with the scree plot presented in (Fig. 1).

Table 5. Principal component of HPLC showing the Eigen values, variability and cumulative variance

	F1	F2	F3	F4	F5	F6	F7	F8	F9
Eigenvalue	4.369	4.051	1.899	1.577	1.451	1.011	0.894	0.658	0.090
Variability (%)	27.306	25.319	11.869	9.858	9.068	6.320	5.587	4.113	0.561
Cumulative %	27.306	52.624	64.493	74.351	83.419	89.739	95.326	99.439	100.000

Fig. 1 [Images not available. See PDF.]

The plot will show the cumulative variance explained by each principal component, allowing you to observe the contributions of PC1 and PC2 to the overall variability

PCA elucidated the factor scores for each significant parameter, revealing that five principal components accounted for a cumulative variability of 83.42%. PC1 displayed the highest eigenvalue (4.369) for axis 1, representing 27.306% of the variability, while PC2 had an eigenvalue of 4.051, signifying 25.319% variability, resulting in a cumulative variability of 52.624%. Naringin, catechol, and quercitin, all with loading coefficients greater than 0.70, primarily contributed to PC1. PC2 was primarily influenced by ferulic acid, luteolin, coumaric acid, veratric acid, and resorcinol, all with loading coefficients greater than 0.60. The percent contribution of PC1 was highest in accession CPM19043 (27.736%), followed by CRR19064 (24.114%) and CRB19004 (23.292%). For PC2, the maximum contribution was reported in accession CRR19064 (30.863%), followed by CRG19007 (25.400%). The score plot of both the first and second principal components collectively explained 52.624% of the total variance (Table S1). Analyzing the biplot based on PCA1 and PCA2, a strong correlation was observed between observations CRG19007 and CRG19013, CRB19004 and CPM19043, CPM19030, CPF19036, and CRR19064, and CPM20070 and CPA20074. However, observation CPM19021 displayed no significant correlation with any other observation. Incorporating the similarity index alongside PCA could prove valuable for effectively classifying C. colocynthis. The grouping of all accessions is detailed in (Fig. 2) providing further insights into their relationships and classifications.

Fig. 2 [Images not available. See PDF.]

Biplot of different phenol and flavonoid standards of methanolic fruit pulp extracts of 10 accessions of Citrullus colocynthis (L.) Schrad

Evaluation of accession on the basis of variation of phenolic and flavonoids by AHC analysis

Cluster 1 includes CRB19004 and CPM19043, primarily characterized by high levels of caffeic acid, resorcinol, vanillin, coumaric acid, and gossypin. CRB19004, positioned at the center of this cluster, exhibits the highest mean value for five different components, with four being phenols and one being a flavonoid (gossypin). In cluster 2, observations CRG19007, CPF19036, CRG19013, and CPA20074 are grouped together, with luteolin (flavonoid) being the predominant characteristic element. CPF19036, collected from Punjab, serves as the central observation in this cluster. Despite their diverse origins, these accessions share similar environmental conditions. Cluster 3 is defined by the presence of coumaric acid and apigenin, including three accessions: CPM19021, CPM19030, and CPM20070. CPM19021 is at the center of this cluster. These accessions were collected from Punjab, showcasing comparable characteristics due to their similar environmental conditions. Cluster 4 comprises accession CRR19064, characterized by eight different phenols and flavonoids: vanillic acid, coumaric acid, ferulic acid, veratric acid, pyrogallol, naringin, catechol, and quercetin (Table S2). These dendrogram clusters represent a distinctive set of accessions with specific phenolic and flavonoid components (Fig. 3).

Fig. 3 [Images not available. See PDF.]

An agglomerative clustering analysis was conducted to reveal clustering patterns based on the variability in concentrations of phenolic and flavonoid components among 10 different accessions. The resulting dendrogram visually demonstrates dissimilarity among the accessions in terms of their phenolic and flavonoid component concentrations. obs1 = CRB19004; obs2 = CRG19007; obs3 = CHR19013; obs4 = CPM19021; obs5 = CPM19030; obs6 = CPB19036; obs7 = CRK19043; obs8 = CPA20064; obs9 = CPM20070; obs10 = CRB20074

Accessions CRB19004 and CPM19043, both originating from Bikaner, Rajasthan, are expected to belong to the same cluster due to their shared regional origin. Similarly, observations CPM19021, CPM19030, and CPM20070, all sourced from Punjab, are likely to cluster together, indicating that plants from the same location share similar elemental composition. This affirms that plants from a specific region possess comparable characteristics. On the other hand, observations CRG19007, CPF19036, CRG19013, and CPA20074, although from diverse locations, exhibit considerable similarities in their regional conditions, aligning them into a single cluster with common prevalent constituents.

Evaluation of accession on the basis of variation of phenolic and flavonoids by Heatmap clustering

A heatmap was generated to comprehensively analyze variations among ten distinct accessions collected from different localities in northern India, focusing on phenol and flavonoid content measurements. Figure 4 presented the data illustrating these parameters for all accessions. The clustering analysis divided the ten accessions into two main groups: Group A and Group B. Group A consisted of only two accessions, CRB19004 and CPM19043, showcasing significant levels of caffeic acid and veratric acid. Group B was further subdivided into two subgroups: Ba and Bb. Subgroup Ba included four accessions CPM20070, CHR19021, CPM19021, and CPM19030 each demonstrating notable amounts of various phytochemicals and showing variability in composition. Subgroup Bb comprised four accessions CRG19013, CPF19036, CRR19064, and CPA20074.

Fig. 4 [Images not available. See PDF.]

A heatmap clustering analysis was performed to illustrate the clustering of 10 different accessions based on varying concentrations of phenols and flavonoids. The top dendrogram represents clustering of metabolites, while the left dendrogram showcases the clustering pattern of accessions. Color intensity ranging from red (highest) to blue (lowest) signifies the levels of phenolic and flavonoid concentrations

In the row dendrogram, constructed based on phenols and flavonoids, the phytochemicals were categorized into two main groups: Group I and Group II. Group I was further divided into subgroups Ia and Ib. Subgroup Ia encompassed six different chemicals-gossypin, coumaric acid, cinnamic acid, luteolin, pyrogallol, and apegenin—exhibiting minimal variability as they were nearly uniform across all studied accessions. Subgroup Ib consisted of four distinct compounds: resorcinol, veratric acid, caffeic acid, and vanillin. Among these, caffeic acid and veratric acid were notably abundant. Group II was also divided into two subgroups: IIa and IIb. Group IIa included two compounds—coumaric acid and ferulic acid—while Group IIb comprised four compounds—vanillic acid, quercetin, naringenin, and catechol. These metabolites exhibited variability and were found in significant amounts across the studied accessions.

Correlation in different accession on the basis of phenols and flavonoids

The correlation matrix provided valuable insights into the relationships and associations among phenols and flavonoids within the studied accessions (Fig. S6). The matrix demonstrated that most phenols exhibited significant correlations, either positive or negative, with other phenolic compounds. For example, ferulic acid showed a strong and positive correlation with coumaric acid and vanillin. Additionally, veratric acid displayed a notable and positive correlation with cinnamic acid and vanillin. Conversely, coumaric acid exhibited a significant negative correlation with cinnamic acid. Concerning flavonoids, the correlation matrix revealed predominantly negative correlations among them. However, there was an exception where quercetin displayed a significant and positive correlation with luteolin and coumaric acid. The correlation matrix elucidated the interconnectedness and interactions among phenolic and flavonoid compounds within the studied accessions, highlighting both positive and negative correlations that play a crucial role in understanding the composition and dynamics of these phytochemicals.

Discussion

The concentrations of total phenolic content in term of TAE/gram of dry weight (DW) of pulp of fruit of C. colocynthis were estimated. Five solvents taken for experimental studies were methanol, ethanol, distilled water, chloroform and petroleum ether. From the current studies it is revealed that maximum amount of phenolics were reported from methanol extract of accession CRR19064 (69.17 ± 0.03 mg TAE/g) followed by extracts of ethanol. While minimum amount was recorded 19.97 ± 0.03 mg TAE/g in accession CPA20074 in chloroform extracts. The content of phenolics in methanol actually varies from the range 69.17 ± 0.03 to 48.43 ± 0.04 in CRR19064 to CPA20074. The content sequence is followed by ethanol with maximum value ranging from 60.37 ± 0.02 in CPM19021 to 43.47 ± 0.03 in CPM20070 as the minimum value. Least amount of phenolic content was reported in CPA20074 with 19.97 ± 0.03mg TAE/g of DW. Similar results to the current study were also reported by Gupta et al., 2018 [17] and Thamer et al., 2023 [18], showing the maximum phenolics content in C. colocynthis pulp extracts.

Phenolics are a broad category of aromatics that contain benzene rings and hydroxyl groups. The component has primary function of protecting plants from herbivorous animals and is even very useful in imparting pharmaceutical properties. Plant phenols also act as antioxidant and free radical scavengers. So, it becomes essential to determine the concentration in plants. In given study the content of flavonoid was also determined and expressed in term of mg quercetin/g in dry weight of pulp. Again, five solvents were used, as mentioned earlier for phenolics, to estimate the amount of flavonoids. Among all five solvents used, maximum amount of flavonoids were reported in methanolic extract followed by ethanol, aqueous, chloroform, and petroleum ether. Among all the accessions, maximum flavonoid content i.e.,76.74 ± 0.03 was found in accession CPM20070. Similarly, Sultan et al., 2010 [19] found 1.39 mg flavonoids, 1.64 mg phenolics compounds and 30.12 mg ascorbic acid per 100 g of entire C. colocynthis plant. Kumar et al., 2008 reported that a methanolic extract of C. colocynthis fruit parts contain 740 mg of Gallic acid equivalent per 100 g plant matter and, as flavonoids, 130 mg of catechin equivalent per 100 g plant matter [20]. HPLC analysis revealed that the quantity of phytochemicals vary significantly in C. colocynthis pulp extracts. Based on total phenolic content (TPC) and total flavonoid content (TFC) screenings, methanol was identified as the best solvent for further investigation. Its high extraction efficiency probably contributes to its applicability for phytochemical studies. Gupta et al., 2018 revealed that methanol extracts of fruit pulp show maximum phytochemical properties and are even responsible for wound healing [21]. The variation in phenolic and flavonoid content among the studied accessions shed light on the species' inherent diversity and its potential implications for bioactivity. Accession-specific variations were noted, with certain accessions displaying notably higher phenolic or flavonoid content than others, emphasizing the significance of genetic factors in determining phytochemical composition. The outcomes of this research indicated that methanolic solvent is the most effective for extraction and it had the maximum levels of phenolics and flavonoids which were diligently related to the highest antioxidant activities. Our findings are consistent with those formerly published studies [20].

The High-Performance Liquid Chromatography (HPLC) analysis provided a detailed insight into the specific phytoconstituents present in the samples. Compounds like caffeic acid, veratric acid, and vanillic acid were found in substantial quantities, contributing significantly to the phenolic profile. In contrast, cinnamic acid, coumaric acid, and pyrogallol were present in minimal amounts. Flavonoid analysis revealed naringin and catechol as the most abundant flavonoids, indicating their potential biological significance [22]. The Principal Component Analysis (PCA) and clustering further illuminated the relationships and classifications among accessions based on their phytochemical profiles. Accessions from the same geographical region tended to cluster together, suggesting a potential influence of environmental factors on the composition of phytochemicals studied previously in some other plants [23]. In summary, this study underscores the importance of solvent selection in phytochemical extraction, highlights the diversity in phenolic and flavonoid composition among C. colocynthis accessions, and provides valuable insights into the specific phytoconstituents present, furthering our understanding of the species and its potential applications in functional foods and pharmaceuticals [24]. In this study, we conducted a comprehensive evaluation of different accessions of C. colocynthis based on the variation in phenolic and flavonoid concentrations using advanced analytical techniques, including Principal Component Analysis (PCA), Agglomerative Hierarchical Clustering (AHC) analysis, and Heat map clustering. These analytical tools allowed us to discern patterns, relationships, and groupings among the accessions.

The PCA analysis was pivotal in understanding the major factors contributing to the variations observed. We found that five principal components accounted for a cumulative variability of 83.42%, with PC1 and PC2 being the most influential. PC1 was primarily associated with phenolic compounds like naringin, catechol, and quercetin, while PC2 was influenced by flavonoids such as luteolin and coumaric acid. The percent contributions of these components varied across different accessions, shedding light on their unique compositions [25]. AHC analysis further reinforced the clustering patterns observed in PCA. It categorized accessions into distinct clusters based on the concentrations of specific phenols and flavonoids. This reaffirmed that geographical origins influence the chemical composition of C. colocynthis, as accessions from the same region tended to share similar characteristics. The heat map clustering provided a visual representation of the phenolic and flavonoid content for each accession, showcasing variations and enabling clear differentiation between groups. The clustering analysis emphasized the presence of specific compounds and revealed their relative abundances in different accessions [26].

Lastly, the correlation matrix illustrated intricate relationships among phenolic and flavonoid compounds, helping us understand their interconnectedness. The presence of both positive and negative correlations among these phytochemicals highlights the complexity of their interactions and their potential role in the biological properties of C. colocynthis. In conclusion, the integration of PCA, AHC, heat map clustering, and correlation analysis allowed for a comprehensive evaluation and characterization of C. colocynthis accessions based on phenolic, flavonoid and antioxidant properties variations. These findings provide valuable insights into the chemical composition and potential applications of this plant species. Future research can build upon this foundation to explore the biological significance and health benefits associated with the observed phenolic and flavonoid profiles.

Conclusion

In conclusion, the wild herbaceous perennial vine, C. colocynthis, presents a promising avenue for drug development. The study demonstrates that this traditionally utilized plant harbors significant potential as a source of novel pharmacological compounds with profound healing properties. The results highlight the methanolic extracts of C. colocynthis as particularly noteworthy, displaying the highest Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) compared to other solvents, underscoring their clinical significance. To harness this potential, it is imperative to focus on the isolation, identification, and separation of bioactive phytochemicals from C. colocynthis. This would not only aid in the formulation of drugs but also facilitate the development of a chemical fingerprint for C. colocynthis cultivars. By comparing the retention time and peak areas of the extracts with established standards, a thorough chemical fingerprinting can be achieved. This analytical approach is critical in identifying cultivars boasting the highest concentrations of bioactive compounds. Despite the known medicinal potential of C. colocynthis in ethnobotanical field, significant biochemical research gaps still remain. Comprehensive photochemical profiling of this plant is still incomplete, leaving many of its principle bioactive compounds (primary and secondary metabolites) and their properties unidentified and uncharacterized. Additionally, while some traditional uses have been documented, systematic photochemical studies are needed to capture the full scope of its applications in different treatment. Furthermore, the response of C. colocynthis to various environmental stresses at the biochemical level is poorly studied, limiting our ability to optimize its cultivation. Addressing these gaps through interdisciplinary research is essential to fully harness the potential of C. colocynthis in medicine and agriculture. Future research on C. colocynthis should focus on several key areas to further unlock its potential for drug development such as isolation and structural interpretation of bioactive compounds, inclusive studies on the pharmacokinetics to understand their mechanisms of action and potential therapeutic effects, and further analysis of bioactive compounds by advance tools of network pharmacology.

Acknowledgements

We are thankful to the Department of Botany, Punjabi University, Patiala (Coordinate, DSA-I of UGC and FIST of DST, New Delhi) for laboratory facilities.

Author contributions

NK: Data Acquisition, Data curation, Writing-Original draft. VG: Writing and editing. CS: Writing and editing. JR: Data curation, Reviewing and editing. MK: Conceptualization, Supervision, Visualization.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

The data supporting the findings of this study are included within the article and its supplementary materials. All relevant datasets generated and analyzed during the current study are available in the supplementary files provided with this publication.

Declarations

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Pandey, MM; Rastogi, S; Rawat, AKS. Indian traditional ayurvedic system of medicine and nutritional supplementation. J Evid Based Complementary Altern Med; 2013; [DOI: https://dx.doi.org/10.1155/2013/376327]

2. Renner, SS; Sousa, A; Chomicki, G. Chromosome numbers, Sudanese wild forms, and classification of the watermelon genus Citrullus, with 50 names allocated to seven biological species. Taxon; 2017; 66, pp. 1393-1405. [DOI: https://dx.doi.org/10.12705/666.7]

3. Dhakad, PK; Sharma, PK; Kumar, S. A review on phytochemical studies and biological potential of Citrullus colocynthis (L.) Schrad (Cucurbitaceae). J Bioeng Biosci; 2017; 5, pp. 55-64. [DOI: https://dx.doi.org/10.13189/bb.2017.050401]

4. Hameed, B; Ali, Q; Hafeez, MM; Malik, A. Antibacterial and antifungal activity of fruit, seed and root extracts of Citrullus colocynthis plant. Biol Clin Sci Res J; 2020; [DOI: https://dx.doi.org/10.54112/bcsrj.v2020i1.33]

5. Joshi SG. Medicinal plants, Oxford and IBH publishing. 2000

6. Meena, MC; Meena, R; Patni, V. High frequency plant regeneration from shoot tip explants of Citrullus colocynthis (Linn.) Schrad.–An important medicinal herb. Afr J Biotechnol; 2010; 9, pp. 5037-5041.

7. da Silva, JAT; Hussain, AI. Citrullus colocynthis (L.) Schrad. (colocynth): biotechnological perspectives. Emir J Food Agric; 2017; [DOI: https://dx.doi.org/10.9755/ejfa.2016-11-1764]

8. Kapoor, M; Kaur, N; Sharma, C; Kaur, G; Kaur, R; Batra, K; Rani, J. Citrullus colocynthis an important plant in indian traditional system of medicine. Pharmacogn Rev; 2020; [DOI: https://dx.doi.org/10.5530/phrev.2020.14.4]

9. Kapoor M, Rani J. Determination of vindoline and rutin content in five different morphotypes of Catharanthus roseus leaves using HPLC. Int J Pharm Pharm Sci. 2019.

10. Singleton, VL; Rossi, JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic; 1965; 16, pp. 144-158. [DOI: https://dx.doi.org/10.5344/ajev.1996.16.3.144]

11. Sanchez-Rangel, JC; Benavides, J; Heredia, JB; Cisneros-Zevallos, L; Jacobo-Velázquez, DA. The Folin-Ciocalteu assay revisited: improvement of its specificity for total phenolic content determination. Anal methods; 2013; 5, pp. 5990-5999. [DOI: https://dx.doi.org/10.1039/C3Y41125G]

12. Aiyegroro, OA; Okoh, AI. Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC. BMC Complement Altern Med; 2010; 10, pp. 1-8.

13. Koleva, II; Van Beek, TA; Linssen, JP; Groot, AD; Evstatieva, LN. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phytochem Anal; 2002; 13, 1 pp. 8-17. [DOI: https://dx.doi.org/10.1002/pca.611]

14. Abdel-Aziz, AWA; Abdel-Motagaly, M; Abdallah, MA; Shaaban, RS; Osman, NS; Elwan, NM. In vitro cytotoxicity, antimicrobial, antioxidant activities and HPLC finger print analyses of the extracts of Ceiba insignis leaves growing in Egypt. Egypt J Chem; 2021; 64, pp. 1831-1843. [DOI: https://dx.doi.org/10.21608/EJCHEM.2021.58716.3267]

15. Hammer, O. PAST: Paleontological statistics software package for education and data analysis. Palaeontrol Electron.; 2001; 4, 2001 9.

16. Lumivero. XLSTAT statistical and data analysis solutions, 2023. https://www.xlstat.com/en.

17. Gupta, SC; Tripathi, T; Paswan, SK; Agarwal, AG; Rao, CV; Sidhu, OP. Phytochemical investigation, antioxidant and wound healing activities of Citrullus colocynthis (bitter apple). Asian Pac J Trop Biomed; 2018; 8, 418. [DOI: https://dx.doi.org/10.4103/2221-1691.239430]

18. Thamer, FH; Thamer, N; Alhamzi, A; Al-Ansi, N; Al-Sadi, S; Al-Shibeh, A. Antioxidant capacity, total phenol contents and phytochemical screening of Citrullus colocynthis crust, pulp and seeds extracts. Ame J Biochem and Biotechnol; 2023; 19, 1 pp. 12-19. [DOI: https://dx.doi.org/10.3844/ajbbsp]

19. Sultan, A; Khan, FU; Iqbal, H; Khan, MA; Khan, IU. Evaluation of chemical analysis profile of Citrullus colocynthis growing in southern areas of Khyber Pukhtunkhwa Pakistan. World Appl Sci J; 2010; 10, pp. 402-405.

20. Kumar, S; Kumar, D; Saroha, K; Singh, N; Vashishta, B. Antioxidant and free radical scavenging potential of Citrullus colocynthis (L.) Schrad. methanolic fruit extract. Acta Pharm; 2008; 58, pp. 215-220. [DOI: https://dx.doi.org/10.2478/v10007-008-0008-1]

21. Behiry, SI; Philip, B; Salem, MZ; Amer, MA; El-Samra, IA; Abdelkhalek, A; Heflish, A. Urtica dioica and Dodonaea viscosa leaf extracts as eco-friendly bioagents against Alternaria alternata isolate TAA-05 from tomato plant. Sci Rep; 2022; 12, 16468. [DOI: https://dx.doi.org/10.1038/s41598-022-20708-4]

22. Kokkini, S; Karousou, R; Vokou, D. Pattern of geographic variations of Origanum vulgare trichomes and essential oil content in Greece. Biochem Syst Ecol; 1994; 22, pp. 517-528. [DOI: https://dx.doi.org/10.1016/0305-1978(94)90046-9]

23. De Rossi, S; Di Marco, G; D'Agostino, A; Braglia, R; Mecca, G; Canini, A; Gismondi, A. Influence of environmental conditions on the production of nutraceuticals in Italian edible plant landraces. Food Res Int; 2023; 165, [DOI: https://dx.doi.org/10.1016/j.foodres.2023.112483]

24. Dhawan, D; Gupta, J. Research article comparison of different solvents for phytochemical extraction potential from datura metel plant leaves. Int J Biol Chem; 2017; 11, 1 pp. 17-22. [DOI: https://dx.doi.org/10.3923/ijbc.2017.17.22]

25. El-Sawi, SA; Ragab, NA; Sleem, AA; Farghaly, AA; Awad, GEA; Maamoun, MAI. Principal component analysis of phenolic compounds of grape waste parts and correlations to their bioassays. Biocatal Agric Biotechnol; 2023; 51, [DOI: https://dx.doi.org/10.1016/j.bcab.2023.102780]

26. AL-Qahtani, SM. Ecophysiological studies of Citrullus colocynthis in response to spatial and seasonal changes in Wadi Al-Akhder, Tabuk Region, Saudi Arabia. Biodiversitas; 2023; [DOI: https://dx.doi.org/10.13057/biodiv/d240623]