Since the start of human civilization, plants have served as sources of food, energy, and medicine due to their safety and non-toxicity, leading to a recent focus on plant based antioxidant compounds. An around 80% of the population in developing countries still relies on traditional medicine, particularly herbal remedies. The plant Diplocyclos palmatus was chosen to quench the demand for a new herbal Medicine (Bannerman Buton & Wen-chieh, 1983).

            Diplocyclos palmatus belongs to the cucurbitaceae family and is commonly referred to as brynoy or lined cucumber. It is indigenous to both rainforests and dry rainforests and is a twining vine with stems that can grow up to 6 meters long. The plant is native to Australia and is often called marble vine (Gupta & Wagh, 2014).

            The Shivalingi plant is a perennial climbing species characterized by a bare stem that thickens and develops pale speckles along the edges, becoming more mature over time.  Leaves are broadly ovate, measuring between 3.5 and 14 cm, and are palmately lobed. The petiole ranges between 1.5 and 9 cm in length.  Flowers are small, pale or yellowish, and appear in clusters of two to eight without stalks, including five female blooms in the same axil. The sepals in the inflorescence measure approximately 3 to 4 mm in length, while those in the female blooms are 1.5 to 2.5 mm long, and the sepals are smaller than the corolla (Gupta & Wagh, 2014).

          Generally, blossom chap is larger than feminine. It has an oblong shape, measuring 1.5 to 2.5 cm when mature, and is scarlet with prominent light stripes. It is native to India and the Himalayas, at altitudes ranging from 200 to 1500 meters. Usually, the blossom period of D. palmatus falls in the autumn season (Gupta & Wagh, 2014). The general composition of Diplocyclos palmatus is given in Table 1

Common Names:

Gujarati              :           Shivalingi

Kannada             :           Lingatonda balli

Hindi                  :           Shivalingi

Malayalam         :           Aiviralikova

Nepal                 :           Shivalingi

Tamil                 :           Aivirali

Telugu               :           Lingadonda

Taxonomic Hierarchy:

          The taxonomic hierarchy of Diplocyclos palmatus (L) C. Jeffrey is as follows:

Kingdom       :           Plantae

Division        :           Angiospermae

Class             :           Dicotyledonae

Order             :          Cucurbitales

Family          :          Cucurbitaceae

Genus            :           Diplocyclos

Species          :           Diplocyclos palmatus (L) C. Jeffrey

Table 1: General composition of Diplocyclos palmatus

                                              (Venkateshwarlu et al., 2011 & Dave et al., 2006)

General constitutions of fatty acids:

          Acid number of D. palmatus is predicted at 2.9%. Iodine value reflects the extent of unsaturation in a fat or oil the D. palmatus showed an iodine number of 171.5. The peroxide value refers to the quantity of peroxide oxygen per 1 kg of fat or oil, which is recorded at 0.3 mEq/kg for D. palmatus. The saponification value indicates the number of milligrams of potassium hydroxide required to saponify 1g of fat under certain conditions. The saponification value of D. palmatus is 208.3 (Venkateshwarlu et al., 2011 & Dave et al., 2006).

Morphological evaluation of D. palmatus:

Stem, leaves, and root:

          D. palmatus plant is a slender vine, with a maximum stem diameter of 2cm. The leaves are deeply palmately lobed, with five prominent, projecting lobes. The size of leaves is approximately 6-13×6-12cm, and the petioles measure about 2-4cm long. Normally, the leaves are crushed to release an unpleasant smell. Also, the upper side of the leaf blade is covered with scattered scab-like hairs. Two extended rings were observed, denoting the leaf's unfavorable attributes (Gupta & Wagh, 2014).  D. palmatus has a tap root system with secondary roots and numerous root hairs. Seldom do fibrous roots arise from the nodes of vines (Thakur & Puri, 2025). Roots have a beneficial effect on asthma (Kranti et al., 2022). Leaf of Diplocyclos palmatus (L.) Jeffrey is depicted in Figure 1.

Figure 1: Leaf of Diplocyclos palmatus (L.) Jeffrey

Flowers:

          Generally, one female flower and three male flowers are found in each leaf axil. The female flowers can grow to 15mm in length, while the male flowers reach about 20mm. Both types of flower petals are 8-10mm long and feature a dense fur covering on their inner surfaces. Each flower contains two anthers, which may be unilocular or bilocular.  The anthers and fibers vary in length from 2-4mm, with a shaggy texture near the base. The hypanthium measures 3-4mm long, and the thick, shaggy staminodes extend towards three stigmas.  Leaf flaps are roughly 2mm long with swollen bases and scattered outgrowths (Gupta & Wagh, 2014). Flower of Diplocyclos palmatus (L.) Jeffrey is represented in Figure 2.

Figure 2: Flower of Diplocyclos palmatus (L.) Jeffrey

Fruits:

           Fruits of Diplocyclos palmatus are ovoid to ellipsoid, measuring around 20-30×15-32mm, and feature random longitudinal markings on their surface.  D. palmatus fruit has an alkaline nature, with a pH of around 8.04 (Venkateshwarlu et al., 2011). In terms of traditional usage, D. palmatus fruit has several medicinal properties and applications.  It’s recognized for its bitter, aperient, and tonic characteristics and is commonly used to relieve bilious ailments, viz., stomach ache, and diarrhoea. Typically, the fruit is used to treat external abscesses (Gupta & Wagh, 2014). In addition, the intake of these fruits has been linked to health complications and mortality in children (Gupta & Wagh, 2014). Likewise, fruits are an effective treatment for chronic colitis (Thakur & Puri, 2025).

Figure 3: Fruit and seeds of Diplocyclos palmatus (L.) Jeffrey

Seedlings:

          Normally, each plant holds nearly 6 to 10 seeds, with each seed roughly 6 to 8 mm long, irregularly shaped, and resembling teardrops.  Cotyledons extend about 4 to 5 mm. The radicle is very small, measuring around 0.8 mm, making it considerably shorter than the cotyledons (Gupta & Wagh, 2014).  An elliptical cotyledon measures about 20-26 X 10-15 mm, and the petioles are almost 2 mm long. Both the petioles and the stalks above the cotyledons are adorned with small, curved trichomes (Gupta & Wagh, 2014).

          The first pair of exact leaves is deeply serrated and tri-lobed. At the end of the tenth leaf, crushed and emit an unpleasant odor.  The margins of the deeply lobed leaves feature 3-5 main lobes that taper into long, narrow points at their tips. The hairs on the stalk are curved and take the place of thorns.  The upper side of the leaf margin is hispid, covered with short hairs (Gupta & Wagh, 2014). Ethanolic and methanolic extracts of D. palmatus seeds exhibit anti-arthritic and anti-diabetic properties (Kadam & Bodhankar, 2013; Tripathi et al., 2012; Jaynarayan et al., 2012). D. palmatus seeds contain 12% essential oil and various proteins, i.e., bryonin, punicic acid (a type of trans fatty acid), goniothalamin, non-ionic glucomannan, and lipids (Singh & Malviya, 2006). Fruit and seeds of Diplocyclos palmatus (L.) Jeffrey is shown in Figure 3.

Qualitative and Quantitative analysis of D. palmatus:

            Alkaloids, flavonoids, triterpenoids, saponins, resins, glycosides, phenolic compounds, and steroids were positive in D. palmatus (Gokulakrishnan et al., 2019). The methanolic extract of D. palmatus leaf and fruit had a higher phenolic content (7.51±0.08 and 9.29±0.01 mg TAE/g extract). In contrast, the aqueous extracts of leaves and fruits showed moderate levels of phenolic compounds, while hexane and chloroform extracts exhibited lower phenolic content (Attar & Ghane, 2017). Phenols are a category of natural secondary metabolites that effectively scavenge free radicals in biological systems.  Increased phenol levels are directly related to enhanced antioxidant activity (Roya & Fatemeh, 2013).

            Tannins are high molecular weight phenolic compounds with various biological functions, including the ability to chelate metal ions and precipitate proteins, and are recognized as effective antioxidants. Reports indicate that tannins possess stronger antioxidant properties than low molecular weight phenolic compounds (Yokozawa et al., 1998). Chloroform extract of D. palmatus leaf (22.07±0.06 mg CE/g) and fruit (6.99±0.10 mg of catechin equivalent/100), the total tannin content exceeded that of carbinol, hexyl hydride, and water extract (Attar & Ghane, 2017).

            Flavonoids are the most common type of antioxidants due to their strong redox potential, acting as singlet oxygen quenchers, reducing agents, hydrogen donors, and metal chelators (Cao et al., 2009). The methanolic extract of D. palmatus fruit has high flavonoids (15.02±0.96 mg CE/g) than the aqueous extract of leaf (9.55±0.65 mg CE/g) (Attar & Ghane, 2017). Terpenoids are the largest and most diverse group of compounds, functioning as growth promoters, influencing fertilization, and acting as anti-feedants (Cao et al., 2009). The methanolic extract of leaves and fruits has the highest concentration of terpenoids (Attar & Ghane, 2017).

Biological activity of Diplocyclos palmatus:

In vitro study of antioxidant activity

            Patel et al. (2020) assayed the ABTS radical on D. palmatus fruit. He concludes that the antioxidant activity is higher than that of ascorbic acid (μg/g).   

 Antimicrobial activity

            Ethanolic extract of D. palmatus exposed to antimicrobial activity against Staphylococcus aureus, Micrococcus luteus, Bacillus cereus, and Pseudomonas aeruginosa by the well diffusion method. Leaf extract of D. palmatus exhibited the highest inhibitory activity against Candida albicans, S. aureus, Shigella, E. coli, and S. typhi. Fruit extract showed good inhibitory effects on S. shigella, Candida albicans, S. typhi, and S. aureus (Gupta & Wagh, 2014).  

Table 2: Biological activities of Diplocyclos palmatus

Anti-cancer activity:

            Fruit extract of D. palmatus had anti-cancer activity against breast (MCF-7) and colon (HT-29) cell lines.  Generally, it’s considered independent of cellular metabolic activity not obstructed by the test compounds. Significant effect of anti-cancer on the MCF-7 (GI50<10, TGI 5.14, LC50 44.27 µg/mL) and HT-29 cell lines (GI50<10, TGI 46.88, LC50 68.31 µg/mL), compared with the standard drug Adriamycin (Alexpandi et al., 2019; Nath et al., 2024 & Manda & Yellu, 2024).  Normally, the combined effects of cucurbitacin B, gemcitabine, docetaxel, methotrexate, and cisplatin are used to treat cancer (Cai, 2015). The efficacy of cucurbitacins B and I in inhibiting the MCF-7 and HT-29 cell lines (Kim et al., 2014 & Gupta & Shrivastava, 2014).    Biological activities of Diplocyclos palmatus are shown in Table 2. 

Anti-diabetic activity:

           D. palmatus plant allowed for various extraction methods, including microwave-assisted extraction, steam bath-assisted extraction, continuous shaking extraction, and ultra-assisted extraction. The microwave-assisted extraction achieved the highest alpha-amylase inhibition (68.68±0.66%). Conversely, steam bath-assisted extraction showed the lowest inhibition (33.52±1.87%). But moderate inhibition was observed in continuous shaking extraction (46.55±1.50%) and ultra-assisted extraction (44.43±0.11%), respectively (Patel et al., 2020).

            Generally, α-glucosidase plays a vital role in postprandial hyperglycemia by hydrolyzing linear and branched isomaltose oligosaccharides, which enhances glucose release. D. palmatus extracts from ultra-assisted extraction exhibited very consistent inhibitory activity (56.27±0.60%). The microwave-assisted extraction method showed lower activity (48.34±0.72%). Similarly, steam bath extraction and continuous shaking extraction have significant inhibitory effects (Zhang, 2011).

Anti-thrombotic activity:

            Usually, thrombosis is considered the formation of a blood clot or the presence of a blood clot within a blood vessel. Ethanolic extract of D. Palmatus was used to treat thrombosis. Results confirmed that D. Palmatus extract controls thrombolytic activity due to phytocomponents found in the plant that can activate plasminogen through fibrin-dependent and fibrin-independent pathways (Gokulakrishnan et al., 2019).   

Anti-pyretic activity & anti-pasmodic effect:

            Kore et al., 2024 found that the alcoholic extract of D. palmatus (L) C. boosts the antispasmodic effect. Over 70% of the alcoholic extract derived from the aerial parts of the D. Palmatus plant exhibited analgesic activity in mice (Ram Kishnan 2019). D. Palmatus fruits are used to control malaria infections (Thakur & Puri, 2025).

Anti-infertility, anti-venom, and antidote activity:  

            Recently, a uterine tonic was developed using D. Palmatus seeds for infertility treatment. D. Palmatus seeds play a significant role in ethnomedicine to treat infertility. This uterine tonic enhances androgenic activity, increases sperm count, raises testosterone levels, stimulates luteinizing hormone, and elevates fructose levels in the seminal vesicles, as measured in serum (Chauhan et al., 2018). The D. Palmatus seed powder (less than 5 grams), mixed with water or milk and consumed regularly for 21 days, aids in regulating the menstrual cycle in women (Gupta & Wagh, 2014). D. Palmatus seeds have anti-inflammatory, antifungal, antimicrobial, analgesic, and anti-hyperlipidemic properties due to the presence of protein (Khan & Khan 2006, Paras et al., 2019, Kamble et al., 2010, Mosaddik et al., 2001, Gowrikumar et al., 1981).   Usually, after a snakebite, fifty grams of D. Palmatus leaf paste mixed with betel leaves should be taken orally three times daily until recovery occurs (Gupta & Wagh, 2014).

Anti -Quorum Sensing activity:

           Methanolic extract of D. Palmatus leaf significantly reduced the biofilm thickness, as confirmed by light microscopy and confocal laser scanning microscopy. Also, reduced the prodigiosin pigment production. In contrast, untreated S. marcescens showed their intricate structure and a dense layer of biofilm cells on glass slides. Nowadays, extracellular virulence in S. marcescens, modulated by quorum sensing, plays a significant role in host infections. Methanolic extract of D. Palmatus leaf significantly reduced the extracellular polysaccharide production against S. marcescens and cell surface hydrophobicity. Decreased protease activity was observed due to tocopherol and phytol in the extract.  Worms exposed to UV-A light, employing the 2',7'-dichlorodihydrofluorescein diacetate stain to evaluate the levels of intracellular reactive oxygen species. Considerably higher ROS level in the UV-A-treated sample than in the control. Extract treated to downregulate ROS levels and improve the lifespan of S. marcescens by hindering bacterial proliferation within the worms (Alexpandi et al., 2019).

          Normally, 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-tetrazolium-5-carboxanilide forms a water soluble colored formazan when it interacts with metabolically active cells. The methanolic extract of D. Palmatus on S. marcescens treated cells exhibited a similar level of formazan as the control.  In contrast, he confirmed that the anti-QS activity of the methanolic extract of D. Palmatus was not due to antibacterial effects (Gowrishankar et al., 2016).

In vivo anti-infection assay:

            Methanolic extract of D. palmatus increased the lifespan of Caenorhabditis elegans by diminishing the virulence of bacterial load within the worms.  A colony forming unit assayed on the extract treated worms had fewer colonies (6 × 10⁴ CFU/mL) than the control (34 × 10⁴ CFU/mL). C. elegans survival assay assessed the efficacy of D. palmatus against S. marcescens. Worms infected with S. marcescens showed complete mortality within 70 hours.  Whereas the extract treated at 200 to 600 μg/mL concentrations, shown to extend the life span by up to five days. 600 μg/mL was recommended for safety and effectiveness (Alexpandi et al., 2019). 

In vitro callus induction activity:

        An efficient in vitro callus stimulation of D. palmatus was achieved from node, internode, and leaf explants on Murashige and Skoog medium with B5 vitamins and different concentrations and combinations of BAP (benzylaminopurine), IAA (indole-3-acetic acid), and IBA (indole-3 Butyric acid). The MS medium formulation containing BAP (1.5 mg/L) + NAA (Napthalene acetic acid) (1 mg/L) + IBA (0.5 mg/L) led to the highest rate of callus induction (Ramar and Ayyadurai, 2015). Regenerated calluses were then transferred to half-strength MS medium fortified with GA3 (1 mg/L) for callus elongation (Upadhyay et al., 2021 & Roopa & Thomas, 2022).

 In silico activity:

          The 3D structure of the SmaR protein was modeled using Phyre2 software. Outcomes of docking analysis showed tocopherol (5.3), β-tocopherol (5.2), tocopherol (5.0), and phytol (3.9) have a higher binding score compared to C4HSL (3.8 K cal/mol) natural ligand (Kelley, 2015). The homology studied on glyceraldehyde-3-phosphate dehydrogenase using D. palmatus; the protein sequences closely match known plant protein sequences (Rubalakshmi, 2020).

Analytical methods of Diplocyclos palmatus:

 FTIR analysis and SEM analysis:

          Fourier transform infrared spectroscopy (FTIR) is primarily used for identifying functional groups. The D. palmatus extract treated and untreated samples showed differences in functional groups. The four key spectral ranges, 3700-3100 cm^-1 (hydration), 3050-2750 cm^-1 (cell membrane fatty acid content), 1700-1500 cm^-1 (amide linkages in proteins and peptides), and 1300-1000 cm^-1 (combined region of proteins and fatty acids), are particularly valuable for evaluating alterations in bacterial cellular mechanisms (Santhakumari et al., 2017).

          The significance of trichome micromorphology in cucurbits was explored using Scanning Electron Microscopy (SEM) across 23 different species (Ali & Alhemaid, 2011). D. palmatus of young and older leaves displayed minimal, poorly developed trichomes with a flattened base at a magnification of X 300.

Chromatographic analysis:

          Fruits of D. palmatus were analyzed by Thin Layer Chromatography (TLC) using various solvents, viz., petroleum ether, chloroform, ethanol, benzene, toluene, ethyl acetate, and aqueous extract. Equal amounts of these extracts were loaded on the TLC plates. After developing the spots, the plates were viewed under ordinary light and UV light (254 nm & 365 nm), and Rf values were calculated. Results proved that the best separation was achieved using benzene as mobile phase (Rf values: 0.14, 0.31, 0.42, 0.50, 0.68, and 0.86), followed by toluene and ethyl acetate in a ratio of 93:7 (Rf values: 0.14, 0.25, 0.51, 0.74, and 0.9), Venkateshwarlu et al., 2011.

          The methanolic extract of D. palmatus was analyzed by gas chromatography-mass spectrometry (GC-MS). Nearly 17 compounds were identified; among them, palmitic acid (78.27%) and phytol (3.14%) were more significant, and octadecadienoic acid (0.23%) had the least area (Alexpandi et al., 2019). The D. palmatus leaf and fruit were analyzed using high performance liquid chromatography to quantify rutin and quercetin. Results showed that the fruits contain 0.0805% rutin and 0.0024% quercitin, respectively. Similarly, leaf had 0.0055% rutin (mg/mL) (Rodge & Biradar, 2016).

          Patel et al., (2020) studied the fruit of D. palmatus using liquid chromatography -mass spectrometry.  Totally, eleven chief compounds were identified. Beta-hederin, a triterpenoid saponin had the highest mass, recorded at 752.491 at m/z 734.45 ([M+H]+). In contrast, Isovaleric acid, a member of the fatty acid group, confirmed the smallest molecular mass at 125.05 g/mol

This review emphasizes that D palmatus has potential antioxidant and anti-thrombotic actions and is especially advantageous for individuals with diabetes. It could also function as a nutraceutical supplement in the creation of various nutrient dense products. Nevertheless, a standardized mechanism is required to isolate and identify its compounds. Additionally, in vivo studies are critical to determine whether these plants interact with pharmaceuticals, including their mechanisms of action, drug absorption, detoxification, interactions, apoptosis, and cell signaling pathways. Further research should be conducted to verify the effectiveness of these approaches in the pharmaceutical and biotechnology industries for developing new drugs targeting antibacterial effects. These drugs are intended to impede the growth of abnormal cells. More research is necessary to assess the product's shelf life.

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